Immune reactivity to her-2/neu protein for diagnosis and treatment of malignancies in which the her-2/neu oncogene is associated

ABSTRACT

Methods for the detection, monitoring and treatment of malignancies in which the HER-2/neu oncogene is associated are disclosed. Detection of specific T cell activation (e.g., by measuring the proliferation of T cells) in response to in vitro exposure to the HER-2/neu protein, or detection of immunocomplexes formed between the HER-2/neu protein and antibodies in body fluid, allows the diagnosis of the presence of a malignancy in which the HER-2/neu oncogene is associated. The present invention also discloses methods and compositions, including peptides, for treating such malignancies.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part application to Ser.No. 106,112, filed Aug. 12, 1993, which is a continuation-in-partapplication to Ser. No. 033,644, filed Mar. 17, 1993, abandoned.

TECHNICAL FIELD

[0002] The present invention is generally directed toward the detection,monitoring, and treatment of malignancies, in which the HER-2/neuoncogene is associated, through the use of a cancer patient's own immunereactivity to the HER-2/neu protein expressed by the HER-2/neu gene.

BACKGROUND OF THE INVENTION

[0003] Despite enormous investments of financial and human resources,cancer remains one of the major causes of death. For example, cancer isthe leading cause of death in women between the ages of 35 and 74.Breast cancer is the most common malignancy in women and the incidencefor developing breast cancer is on the rise. One in nine women will bediagnosed with the disease. Standard approaches to cure breast cancerhave centered around a combination of surgery, radiation andchemotherapy. These approaches have resulted in some dramatic successesin certain malignancies. However, breast cancer is most often incurable,when diagnosed beyond a certain stage. Alternative approaches to earlydiagnosis and therapy are necessary.

[0004] A common characteristic of malignancies is uncontrolled cellgrowth. Cancer cells appear to have undergone a process oftransformation from the normal phenotype to a malignant phenotypecapable of autonomous growth. Amplification and overexpression ofsomatic cell genes is considered to be a common primary event thatresults in the transformation of normal cells to malignant cells. Themalignant phenotypic characteristics encoded by the oncogenic genes arepassed on during cell division to the progeny of the transformed cells.

[0005] Ongoing research involving oncogenes has identified at leastforty oncogenes operative in malignant cells and responsible for, orassociated with, transformation. Oncogenes have been classified intodifferent groups based on the putative function or location of theirgene products (such as the protein expressed by the oncogene).

[0006] Oncogenes are believed to be essential for certain aspects ofnormal cellular physiology. In this regard, the HER-2/neu oncogene is amember of the tyrosine protein kinase family of oncogenes and shares ahigh degree of homology with the epidermal growth factor receptor.HER-2/neu presumably plays a role in cell growth and/or differentiation.HER-2/neu appears to induce malignancies through quantitative mechanismsthat result from increased or deregulated expression of an essentiallynormal gene product.

[0007] HER-2/neu (p185) is the protein product of the HER-2/neuoncogene. The HER-2/neu gene is amplified and the HER-2/neu protein isoverexpressed in a variety of cancers including breast, ovarian, colon,lung and prostate cancer. HER-2/neu is related to malignanttransformation. It is found in 50%-60% of ductal in situ carcinoma and20%-40% of all breast cancers, as well as a substantial fraction ofadenocarcinomas arising in the ovaries, prostate, colon and lung.HER-2/neu is intimately associated not only with the malignantphenotype, but also with the aggressiveness of the malignancy, beingfound in one-fourth of all invasive breast cancers. HER-2/neuoverexpression is correlated with a poor prognosis in both breast andovarian cancer. HER-2/neu is a transmembrane protein with a relativemolecular mass of 185 kd that is approximately 1255 amino acids (aa) inlength. It has an extracellular binding domain (ECD) of approximately645 aa, with 40% homology to epidermal growth factor receptor (EGFR), ahighly hydrophobic transmembrane anchor domain (TMD), and acarboxyterminal cytoplasmic domain (CD) of approximately 580 aa with 80%homology to EGFR.

[0008] An approach to developing a diagnostic assay for malignancies, inwhich the HER-2/neu oncogene is associated, has been to attempt toquantify the protein expression product of the HER-2/neu oncogene intissue or body fluids. However, there have been problems in thedevelopment of diagnostic assays based on direct detection of HER-2/neuprotein.

[0009] Due to the difficulties in the current approaches to diagnosisand therapy of cancers in which the HER-2/neu oncogene is associated,there is a need in the art for improved methods and compositions. Thepresent invention fills this need, and further provides other relatedadvantages.

SUMMARY OF THE INVENTION

[0010] Briefly stated, the present invention provides a variety ofmethods for the detection of a malignancy in a warm-blooded animal,wherein a HER-2/neu oncogene is associated with the malignancy. Themethods may be used on a one time basis when a malignancy is suspectedor on a periodic basis, e.g., to monitor an individual with an elevatedrisk of acquiring or reacquiring a malignancy. In one embodiment, themethod comprises the steps of: (a) isolating CD4⁺ T cells from awarm-blooded animal; (b) incubating the T cells with HER-2/neu protein;and (c) detecting the presence or absence of specific activation of theT cells, thereby determining the presence or absence of the malignancy.In another embodiment, the method comprises the steps of: (a) isolatingCD8⁺ T cells from a warm-blooded animal; (b) incubating the T cells withHER-2/neu protein; and (c) detecting the presence or absence of specificactivation of the T cells, thereby determining the presence or absenceof the malignancy. In another embodiment, the method comprises the stepsof: (a) contacting a body fluid, suspected of containing antibodiesspecific for HER-2/neu protein, with HER-2/neu protein; (b) incubatingthe body fluid under conditions and for a time sufficient to allowimmunocomplexes to form; and (c) detecting the presence or absence ofimmunocomplexes formed between the HER-2/neu protein and antibodies inthe body fluid specific for the HER-2/neu protein, thereby determiningthe presence or absence of the malignancy.

[0011] In another aspect, the present invention provides methods formonitoring the effectiveness of cancer therapy in a warm-blooded animalwith a malignancy, wherein a HER-2/neu oncogene is associated with themalignancy. Uses of such methods include the early detection of relapse.In one embodiment, the method comprises the steps of: (a) contacting afirst body fluid sample, taken from the warm-blooded animal prior toinitiation of therapy, with HER-2/neu protein; (b) incubating the bodyfluid under conditions and for a time sufficient to allowimmunocomplexes to form; (c) detecting immunocomplexes formed betweenthe HER-2/neu protein and antibodies in the body fluid specific for theHER-2/neu protein; (d) repeating steps (a), (b), and (c) on a secondbody fluid sample taken from the animal subsequent to the initiation oftherapy; and (e) comparing the number of immunocomplexes detected in thefirst and second body fluid samples, thereby monitoring theeffectiveness of the therapy in the animal.

[0012] The present invention is also directed toward methods fortreating a malignancy in a warm-blooded animal, wherein a HER-2/neuoncogene is associated with the malignancy. In one embodiment, themethod comprises the steps of: (a) isolating CD4⁺ T cells from awarm-blooded animal; (b) incubating the T cells in the presence ofHER-2/neu protein, such that the T cells proliferate; and (c)administering to the warm-blooded animal an effective amount of theproliferated T cells. In another embodiment, the method comprises thesteps of: (a) isolating CD8⁺ T cells from a warm-blooded animal; (b)incubating the T cells in the presence of HER-2/neu protein, such thatthe T cells proliferate; and (c) administering to the warm-bloodedanimal an effective amount of the proliferated T cells. In anotherembodiment, the method comprises the steps of: (a) isolating CD4⁺T cellsfrom a warm-blooded animal; (b) incubating the T cells in the presenceof HER-2/neu protein, such that the T cells proliferate; (c) cloning oneor more cells that proliferated in the presence of HER-2/neu protein;and (d) administering to the warm-blooded animal an effective amount ofthe cloned T cells. In another embodiment, the method comprises thesteps of: (a) isolating CD8⁺ T cells from a warm-blooded animal; (b)incubating the T cells in the presence of HER-2/neu protein, such thatthe T cells proliferate; (c) cloning one or more cells that proliferatedin the presence of HER-2/neu protein; and (d) administering to thewarm-blooded animal an effective amount of the cloned T cells. In yetanother embodiment, the method comprises immunizing the animal with aHER-2/neu peptide recognized by T cells, the peptide not being theextracellular domain of the protein expression product of a HER-2/neuoncogene.

[0013] Within a related aspect, the present invention providesanti-cancer therapeutic compositions comprising T cells proliferated inthe presence of HER-2/neu protein, in combination with apharmaceutically acceptable carrier or diluent. In addition, a varietyof peptides designated for CD8⁺ T cell responses are provided whichinclude peptides consisting essentially of:

[0014] His-Leu-Tyr-Gln-Gly-Cys-Gln-Val-Val (Seq. ID No. 1);

[0015] Pro-Leu-Gln-Pro-Glu-Gln-Leu-Gln-Val (Seq. ID No. 2);

[0016] Pro-Leu-Thr-Ser-Ile-Ile-Ser-Ala-Val (Seq. ID No. 3);

[0017] Ile-Leu-Leu-Val-Val-Val-Leu-Gly-Val (Seq. ID No. 4);

[0018] Leu-Leu-Val-Val-Val-Leu-Gly-Val-Val (Seq. ID No. 5);

[0019] Arg-Leu-Leu-Gln-Glu-Thr-Glu-Leu-Val (Seq. ID No. 6);

[0020] Cys-Leu-Thr-Ser-Thr-Val-Gln-Leu-Val (Seq. ID No. 7);

[0021] Asp-Leu-Ala-Ala-Arg-Asn-Val-Leu-Val (Seq. ID No. 8);

[0022] Val-Leu-Val-Lys-Ser-Pro-Asn-His-Val (Seq. ID No. 9);

[0023] Thr-Leu-Ser-Pro-Gly-Lys-Asn-Gly-Val (Seq. ID No. 10);

[0024] Val-Leu-Gly-Val-Val-Phe-Gly-Ile-Leu (Seq. ID No. 11);

[0025] Leu-Ile-Lys-Arg-Arg-Gln-Gln-Lys-Ile (Seq. ID No. 12);

[0026] Lys-Ile-Pro-Val-Ala-Ile-Lys-Val-Leu (Seq. ID No. 13);

[0027] Ile-Leu-Asp-Glu-Ala-Tyr-Val-Met-Ala (Seq. ID No. 14);

[0028] Gln-Leu-Met-Pro-Tyr-Gly-Cys-Leu-Leu (Seq. ID No. 15);

[0029] Gln-Ile-Ala-Lys-Gly-Met-Ser-Tyr-Leu (Seq. ID No. 16);

[0030] Leu-Leu-Asn-Trp-Cys-Met-Gln-Ile-Ala (Seq. ID No. 17);

[0031] Arg-Leu-Val-His-Arg-Asp-Leu-Ala-Ala (Seq. ID No. 18);

[0032] Asp-Ile-Asp-Glu-Thr-Glu-Tyr-His-Ala (Seq. ID No. 19);

[0033] Asp-Leu-Leu-Glu-Lys-Gly-Glu-Arg-Leu (Seq. ID No. 20);

[0034] Thr-Ile-Asp-Val-Tyr-Met-Leu-Met-Val (Seq. ID No. 21);

[0035] Met-Ile-Met-Val-Lys-Cys-Trp-Met-Ile (Seq. ID No. 22);

[0036] Asp-Leu-Val-Asp-Ala-Glu-Glu-Tyr-Leu (Seq. ID No. 23);

[0037] Gly-Leu-Glu-Pro-Ser-Glu-Glu-Glu-Ala (Seq. ID No. 24); or

[0038] Tyr-Leu-Thr-Pro-Gln-Gly-Gly-Ala-Ala (Seq. ID No. 25).

[0039] Similarly, a variety of peptides designated for CD4⁺ T cellresponses are provided which include peptides consisting essentially of:

[0040] His-Leu-Asp-Met-Leu-Arg-His-Leu-Tyr-Gln-Gly-Cys-Gln-Val-Val (Seq.ID No. 30);

[0041] Pro-Leu-Gln-Arg-Leu-Arg-Ile-Val-Arg-Gly-Thr-Gln-Leu-Phe-Glu (Seq.ID No. 31);

[0042] Leu-Arg-Ser-Leu-Thr-Glu-Ile-Leu-Lys-Gly-Gly-Val-Leu-Ile-Gln (Seq.ID No. 32);

[0043] Val-Thr-Tyr-Asn-Thr-Asp-Thr-Phe-Glu-Ser-Met-Pro-Asn-Pro-Glu (Seq.ID No. 33);

[0044] His-Leu-Arg-Glu-Val-Arg-Ala-Val-Thr-Ser-Ala-Asn-Ile-Gln-Glu (Seq.ID No. 34);

[0045] Val-Arg-Ala-Val-Thr-Ser-Ala-Asn-Ile-Gln-Glu-Phe-Ala-Gly-Cys (Seq.ID No. 35);

[0046] Asn-Ile-Gln-Glu-Phe-Ala-Gly-Cys-Lys-Lys-Ile-Phe-Gly-Ser-Leu (Seq.ID No. 36);

[0047] Gln-Val-Phe-Glu-Thr-Leu-Glu-Glu-Ile-Thr-Gly-Tyr-Leu-Tyr-Ile (Seq.ID No. 37);

[0048] Gln-Glu-Cys-Val-Glu-Glu-Cys-Arg-Val-Leu-Gln-Gly-Leu-Pro-Arg (Seq.ID No. 38);

[0049] Val-Val-Val-Leu-Gly-Val-Val-Phe-Gly-Ile-Leu-Ile-Lys-Arg-Arg (Seq.ID No. 39);

[0050] Lys-Tyr-Thr-Met-Arg-Arg-Leu-Leu-Gln-Glu-Thr-Glu-Leu-Val-Glu (Seq.ID No. 40);

[0051] Gly-Ala-Met-Pro-Asn-Gln-Ala-Gln-Met-Arg-Ile-Leu-Lys-Glu-Thr (Seq.ID No. 41);

[0052] Val-Lys-Val-Leu-Gly-Ser-Gly-Ala-Phe-Gly-Thr-Val-Tyr-Lys-Gly (Seq.ID No. 42);

[0053] Ser-Pro-Lys-Ala-Asn-Lys-Glu-Ile-Leu-Asp-Glu-Ala-Tyr-Val-Met (Seq.ID No. 43);

[0054] Gly-Val-Gly-Ser-Pro-Tyr-Val-Ser-Arg-Leu-Leu-Gly-Ile-Cys-Leu (Seq.ID No. 44);

[0055] Ser-Arg-Leu-Leu-Gly-Ile-Cys-Leu-Thr-Ser-Thr-Val-Gln-Leu-Val (Seq.ID No.-45);

[0056] Gly-Ser-Gln-Asp-Leu-Leu-Asn-Trp-Cys-Met-Gln-Ile-Ala-Lys-Gly (Seq.ID No. 46);

[0057] Val-Lys-Ile-Thr-Asp-Phe-Gly-Leu-Ala-Arg-Leu-Leu-Asp-Ile-Asp (Seq.ID No. 47);

[0058] Thr-Val-Trp-Glu-Leu-Met-Thr-Phe-Gly-Ala-Lys-Pro-Tyr-Asp-Gly (Seq.ID No. 48);

[0059] Pro-Ala-Arg-Glu-Ile-Pro-Asp-Leu-Leu-Glu-Lys-Gly-Glu-Arg-Leu (Seq.ID No. 49);

[0060] Arg-Phe-Arg-Glu-Leu-Val-Ser-Glu-Phe-Ser-Arg-Met-Ala-Arg-Asp (Seq.ID No. 50);

[0061] Glu-Asp-Asp-Asp-Met-Gly-Asp-Leu-Val-Asp-Ala-Glu-Glu-Tyr-Leu (Seq.ID No. 51);

[0062] Gly-Met-Gly-Ala-Ala-Lys-Gly-Leu-Gln-Ser-Leu-Pro-Thr-His-Asp (Seq.ID No. 52);

[0063] Thr-Cys-Ser-Pro-Gln-Pro-Glu-Tyr-Val-Asn-Gln-Pro-Asp-Val-Arg (Seq.ID No. 53);

[0064] Thr-Leu-Glu-Arg-Pro-Lys-Thr-Leu-Ser-Pro-Gly-Lys-Asn-Gly-Val (Seq.ID No. 54);

[0065] Gly-Gly-Ala-Val-Glu-Asn-Pro-Glu-Tyr-Leu-Thr-Pro-Gln-Gly-Gly (Seq.ID No. 55);

[0066] Asn-Gln-Glu-Val-Thr-Ala-Glu-Asp-Gly-Thr-Gln-Arg-Cys-Glu-Lys (Seq.ID No. 56);

[0067] Gln-Val-Ile-Arg-Gly-Arg-Ile-Leu-His-Asn-Gly-Ala-Tyr-Ser-Leu (Seq.ID No. 57);

[0068] Leu-Gln-Val-Phe-Glu-Thr-Leu-Glu-Glu-Ile-Thr-Gly-Tyr-Leu-Tyr (Seq.ID No. 58);

[0069] Ala-Ser-Pro-Leu-Thr-Ser-Ile-Ile-Ser-Ala-Val-Val-Gly-Ile-Leu (Seq.ID No. 59);

[0070]Thr-Gln-Arg-Cys-Glu-Lys-Cys-Ser-Lys-Pro-Cys-Ala-Arg-Val-Cys-Tyr-Gly-Leu(Seq. ID No. 60);

[0071]Arg-Leu-Arg-Ile-Val-Arg-Gly-Thr-Gln-Leu-Phe-Glu-Asp-Asn-Tyr-Ala-Leu(Seq. ID No. 61);

[0072] Lys-Ile-Phe-Gly-Ser-Leu-Ala-Phe-Leu-Pro-Glu-Ser-Phe-Asp-Gly-Asp(Seq. ID No. 62);

[0073] Arg-Arg-Leu-Leu-Gln-Glu-Thr-Glu-Leu-Val-Glu-Pro-Leu-Thr-Pro-Ser(Seq. ID No. 63); or

[0074] Glu-Leu-Val-Ser-Glu-Phe-Ser-Arg-Met-Ala-Arg-Asp-Pro-Gln (Seq. IDNo. 64).

[0075] Additional peptides are provided and include a peptide consistingessentially of the amino acid sequence of FIG. 1 from lysine, amino acid676, to valine, amino acid 1255.

[0076] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0077]FIG. 1 shows that p185^(HER-2/neu) protein contains multiplesegments with amino acid sequences appropriate for binding to class IIMHC molecules. Each outlined amino acid represents the center point ofan 11-mer peptide with alpha-helical periodicity and amphipathicity.Each underlined amino acid segment represents an epitope correspondingto Rothbard and Taylor motifs.

[0078]FIG. 2 graphically illustrates the results of a low frequencyevent screening from a normal individual indicating that a CD4⁺ T cellresponse can be detected against p185^(HER-2/neu) and peptides derivedfrom its amino acid sequence. The graph represents the data from onenormal individual analyzed with the low frequency screening assayeddescribed further below. Positive responses to the intact protein andtwo peptides were detected.

[0079]FIG. 3 (panels A-D) graphically illustrates that CD4⁺ T cellsreactive to p185^(HER-2/neu) protein and peptides can be detected inhigh frequency from patients with HER-2/neu positive breast cancer andcan also be detected in some patients with tumors that test negativelyfor expression of p185^(HER-2/neu) protein. All four breast cancerpatients represented here, patient A (panel A), patient B (panel B),patient C (panel C), and patient D (panel D), were premenopausal women.Patient A had a primary tumor that tested negatively for overexpressionof p185^(HER-2/neu). The other three patients had HER-2/neu positivetumors. A proliferation assay was performed using purified peripheralblood mononuclear cells (PBMC) as described below, with eachexperimental group done in 24 well replicate. Two×10⁵ PBMC/well wereincubated with no antigen, tetanus toxoid (5 μg/ml), p185^(HER-2/neu) (5μg/ml), or HER-2/neu derived peptides (50 μg/ml) as described furtherbelow. After 4 days, wells were pulsed with 1 μCi of tritiated thymidine(³H-TdR) for 6-8 hours and then counted. The data represents the mean of24 determinations of the c.p.m. with standard error bars expressed.

[0080]FIG. 4 (panels A and B) graphically illustrates that CD8⁺ CTLspecific for HER-2/neu peptides 48-56 and 789-797 can be generated by invitro immunization. Three×10⁷ PBMC from a homozygous HLA-A2 normal donorwere incubated with p48-56 or p789-797^(HER-2/neu) peptides atconcentrations of 10 μg/ml. The lymphocytes were tested for lyticactivity after 10 in vitro sensitizations (IVS). Data is depicted afterthe tenth IVS with p48-56 (panel A) or with p789-797 (panel B). Targetcells consisted of ⁵¹Cr-labeled autologous EBV transformed B lymphocyteswhich had been incubated with p48-56 or p789-797^(HER-2/neu) or anirrelevant peptide for 2 hours prior to use. A four hour chromiumrelease assay (CRA) was performed. The results represent the percentspecific lysis at the indicated effector:target (E:T) ratio. Targetcontrols of ⁵¹Cr-labeled K562 and Daudi cells were also included toevaluate NK and LAK activity. The execution of the CRA is as described.The results represent the percent specific lysis at the indicatedeffector:target (E:T) ratio.

[0081]FIG. 5 pictorially illustrates that antibodies are detectableagainst p185^(HER-2/neu) in the sera of a breast cancer patient. Lane 1represents the immunoblot of p185 using a HER-2/neu positive breastcancer patient's sera (1:1000 dilution) as primary antibody. The blotwas analyzed as described further below. Lane 2 represents the controlstrip from that experiment developed with c-neu antibody.

[0082]FIG. 6 pictorially shows that antibodies in the sera of a breastcancer patient identify the same p185 band as does a knownHER-2/neu-specific antibody (“control antibody”). A membrane preparationfrom NIH3T3 cells (a murine cell line) that had been transfected withHER-2/neu cDNA (“NIH3T3+H2N”) was tested against control antibody (laneA) or patient sera (lane D). Similarly, a membrane preparation fromuntransfected cells (“NIH3T3”) was tested against control antibody (laneB) or patient sera (lane C).

[0083]FIG. 7 pictorially illustrates that some breast cancer patientshave antibodies directed to both the extracellular and intracellulardomain of the HER-2/neu protein. Sera of breast cancer patients istested against the extracellular domain (“ECD protein”) or theintracellular domain (“ICD protein”), in lanes A and B, respectively.

[0084]FIG. 8 graphically illustrates that rats immunized with peptidesderived from the intracellular domain (ICD) portion of rat neu proteindevelop antibody responses to neu protein. An ELISA was performed toevaluate peptide immunized animals for antibody responses tonon-transforming rat neu protein. Each sera was analyzed at a 1:25,1:50, 1:100, and 1:200 dilution. The OD value shown is that of thebackground wells subtracted from the wells coated with neu protein. Alldata shown is at a rat sera concentration of 1:25. Control sera wasderived from an animal immunized with adjuvant alone. Antibody responsestitered with decreasing serum concentrations. Results were reproduciblein 3 separately run assays.

[0085]FIG. 9 graphically illustrates that rats immunized with peptidesderived from the extracellular domain (ECD) portion of rat neu proteindevelop antibody responses to neu protein. ELISA evaluation wasperformed as described in FIG. 8. All data shown is at a rat seraconcentration of 1:25. Control sera was derived from an animal immunizedwith adjuvant alone. Antibody responses titered with decreasing serumconcentrations. Results were reproducible in 3 separately run assays.

[0086]FIG. 10 graphically shows that epitope analysis of ICD antibodyresponses demonstrates dominant B cell epitopes as well as “determinantspreading” between domains. ELISA analysis for peptide epitopes wasperformed. Each animal's sera was evaluated at dilutions of 1:25, 1:50,1:100, and 1:200-or each peptide analyzed. Antibody responses titeredwith decreasing serum concentrations. All data shown is at a rat seraconcentration of 1:50. Control sera analyzed was pooled sera from 5non-immunized animals. Results were reproducible in 3 separately runassays.

[0087]FIG. 11 graphically shows that epitope analysis of ECD antibodyresponses demonstrates dominant B cell epitopes. ELISA analysis forpeptide epitopes was performed. Each animal's sera was evaluated atdilutions of 1:25, 1:50, 1:100, and 1:200 for each peptide analyzed.Antibody responses titered with decreasing serum concentrations. Alldata shown is at a rat sera concentration of 1:50. Control sera analyzedwas pooled sera from 5 non-immunized animals. Results were reproduciblein 3 separately run assays.

[0088]FIG. 12 pictorially illustrates that antibodies elicited byimmunization to either ICD or ECD peptides are specific for and canimmunoprecipitate both rat neu protein and human HER-2/neu protein.Panel A shows the results of an immunoprecipitation experiment withimmunized rat sera and lysates of DHFRG-8. Each sera was able toimmunoprecipitate rat neu from the cell lysates. The immunoprecipitateswere resolved on a 7.5% SDS-acrylamide gel and transferred tonitrocellulose. The blots were probed with primary antibody, c-neu-Ab-3,at a 1:1000 dilution. Control sera of an animal immunized with theadjuvant alone showed no evidence of reactivity to rat neu. Panel Bdepicts the results of an immunoprecipitation experiment with immunizedrat sera and lysates of SKBR3, a source of human neu. Immunoblotting wasperformed in an identical manner and all experimental animal sera wereable to immunoprecipitate human neu. The control sera, again, showed noevidence of reactivity.

[0089]FIG. 13 pictorially illustrates that B cell epitopes that arecross reactive between human and rat neu are present in both domains ofthe protein. Shown here are the results of Western blot analysis ofprotein domain epitope mapping from representative animals in eachimmunized group. Animal 1.2 was immunized with the ECD group ofpeptides, animal 2.2 with the ICD group of peptides, and the controlanimal was immunized with adjuvant alone. Proteins were electrophoresed.After transfer to nitrocellulose the blots were incubated for 18 hoursin rat sera at a 1:500 dilution. Antibody responses were detected with asecond step goat anti-rat Ig HRP antibody at a dilution of 1:5000.Responses were detected to both human and rat neu as well as to bothhuman ICD and ECD domain recombinant proteins. Antibody responses tothese proteins could not be detected in the control animal which wasimmunized with adjuvant alone. Although data are shown here for animals1.2, 2.2, and control, all animals in each group had the same pattern ofresponse.

[0090]FIG. 14 graphically illustrates that immunization of rats with ICDpeptides elicits neu peptide-specific T cell responses. 2×10⁵ immunizedspleen cells were incubated with 25 μg/ml of the various peptides. The“Mix” group consisted of 25 μg/ml each of the immunizing peptides. Aproliferation assay was performed. Each experimental group was done in 6well replicates. The data is expressed in terms of a stimulation index(SI) which is the mean of the experimental wells divided by the mean ofthe control (no antigen) wells. Stimulation indices greater than 2 areconsidered to be indicative of a primed response. Animals immunized withadjuvant alone showed no stimulation index greater than 0.9 to any ofthe tested peptides (data not shown).

[0091]FIG. 15 graphically illustrates that immunization of rats with ICDpeptides elicits neu protein-specific T cell responses. 1×10⁵ cultured Tcells derived from immunized spleen were incubated with 1×10⁵ syngeneicspleen as APC (antigen presenting cells) and 1 μg/ml of purified rat neuprotein. Each experimental group was done in 6 well replicates. The datais expressed in terms of a stimulation index which is the mean of theexperimental wells divided by the mean of the control (no antigen)wells. Stimulation indices greater than 2 are considered to beindicative of a primed response. Wild type ras protein was theirrelevant protein used in the assay.

[0092]FIG. 16 graphically shows that immunization of rats with ECDpeptides elicits only weak peptide-specific T cell responses. 2×10⁵immunized spleen cells were incubated with 25 μg/ml of the variouspeptides. The “Mix” group consisted of 25 μg/ml each of the immunizingpeptides. Each experimental group was done in 6 well replicates. Thedata is expressed in terms of a stimulation index which is the mean ofthe experimental wells divided by the mean of the control (no antigen)wells. Stimulation indices greater than 2 are considered to beindicative of a primed response. Animals immunized with adjuvant aloneshowed no stimulation index greater than 1.0 to any of the testedpeptides (data not shown).

[0093]FIG. 17 graphically shows that immunization of rats with ECDpeptides elicits weak, but positive, responses to neu protein. 1×10⁵cultured T cells derived from immunized spleen or lymph nodes wereincubated with 1×10⁵ syngeneic spleen as APC and 1 μg/ml of purified ratneu protein. Each experimental group was done in 6 well replicates. Thedata is expressed in terms of a stimulation index which is the mean ofthe experimental wells divided by the mean of the control (no antigen)wells. Stimulation indices greater than 2 are considered to beindicative of a primed response. Wild type ras protein was theirrelevant protein used in the assay.

DETAILED DESCRIPTION OF THE INVENTION

[0094] Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter.

[0095] HER-2/neu Protein—as used herein, refers to the p185 protein(also known as c-erbB2) and peptides thereof which are recognized byhelper T cells or cytotoxic T cells; and may be naturally derived,synthetically produced, genetically engineered, or a functionalequivalent thereof, e.g., where one or more amino acids are replaced byother amino acid(s) or non-amino acid(s) which do not substantiallyaffect function.

[0096] Proliferation of T cells—as used herein, includes themultiplication of T cells as well as the stimulation of T cells leadingto multiplication, i.e., the initiation of events leading to mitosis andmitosis itself. Methods for detecting proliferation of T cells arediscussed below.

[0097] As noted above, the present invention is directed toward methodsand compositions for the diagnosis, monitoring and treatment ofmalignancies in a warm-blooded animal, wherein an amplified HER-2/neugene is associated with the malignancies. Association of an amplifiedHER-2/neu gene with a malignancy does not require that the proteinexpression product of the gene be present on the tumor. For example,overexpression of the protein expression product may be involved withinitiation of a tumor, but the protein expression may subsequently belost. An effective autochthonous immune response may convert a HER-2/neupositive tumor to HER-2/neu negative, but existent immunity will bepresent and allow diagnosis.

[0098] More specifically, the disclosure of the present invention, inone aspect, shows that the protein expression product of the HER-2/neugene can be recognized by thymus-dependent lymphocytes (hereinafter “Tcells”) and, therefore, the autochthonous immune T cell response can beutilized to diagnose, monitor and treat malignancies in which such aprotein is or has been overexpressed. The disclosure of the presentinvention also shows, in another aspect, that sera of patients with amalignancy, in which an amplified HER-2/neu oncogene is associated,contain antibodies to HER-2/neu protein. The autochthonous antibodyresponse can be used to diagnose, monitor and treat malignancies inwhich such a protein is overexpressed.

[0099] It is well known that the two major arms of the immune systemare: (1) cell-mediated immunity with immune T cells and (2) humoralimmunity with antibodies. Further, the immune system normally functionsto recognize and destroy any foreign or aberrant cells in the body.Since the HER-2/neu protein is expressed by some normal cells, toleranceand/or anergy (i.e., diminished reactivity to a specific antigen) isexpected. Thus, it is surprising that, as disclosed within the presentinvention, both T cell and antibody responses to HER-2/neu are detected.

[0100] In general, CD4⁺ T cell populations are considered to function ashelpers/inducers through the release of lymphokines when stimulated by aspecific antigen; however, a subset of CD4⁺ cells can act as cytotoxic Tlymphocytes (CTL). Similarly, CD8⁺ T cells are considered to function bydirectly lysing antigenic targets; however, under a variety ofcircumstances they can secrete lymphokines to provide helper or DTHfunction. Despite the potential of overlapping function, the phenotypicCD4 and CD8 markers are linked to the recognition of peptides bound toclass II or class I MHC antigens. The recognition of antigen in thecontext of class II or class I MHC mandates that CD4⁺ and CD8⁺ T cellsrespond to different antigens or the same antigen presented underdifferent circumstances. The binding of immunogenic peptides to class IIMHC antigens most commonly occurs for antigens ingested by antigenpresenting cells. Therefore, CD4⁺ T cells generally recognize antigensthat have been external to the tumor cells. By contrast, under normalcircumstances, binding of peptides to class I MHC occurs only forproteins present in the cytosol and synthesized by the target itself,proteins in the external environment are excluded. An exception to thisis the binding of exogenous peptides with a precise class I bindingmotif which are present outside the cell in high concentration. Thus,CD4⁺ and CD8⁺ T cells have broadly different functions and tend torecognize different antigens as a reflection of where the antigensnormally reside.

[0101] As disclosed within the present invention, the protein productexpressed by the HER-2/neu oncogene is recognized by T cells. Such aprotein expression product “turns over” within cells, i.e., undergoes acycle wherein a synthesized protein functions and then eventually isdegraded and replaced by a newly synthesized molecule. During theprotein life cycle, peptide fragments from the protein bind to majorhistocompatibility complex (MHC) antigens. By display of a peptide boundto MHC antigen on the cell surface and recognition by host T cells ofthe combination of peptide plus self MHC antigen, a malignant cell willbe immunogenic to T cells. The exquisite specificity of the T cellreceptor enables individual T cells to discriminate between proteinfragments which differ by a single amino acid residue.

[0102] During the immune response to a peptide, T cells expressing a Tcell receptor with high affinity binding of the peptide-MHC complex willbind to the peptide-MHC complex and thereby become activated and inducedto proliferate. In the first encounter with a peptide, small numbers ofimmune T cells will secrete lymphokines, proliferate and differentiateinto effector and memory T cells. The primary immune response will occurin vivo but has been difficult to detect in vitro. Subsequent encounterwith the same antigen by the memory T cell will lead to a faster andmore intense immune response. The secondary response will occur eitherin vivo or in vitro. The in vitro response is easily gauged by measuringthe degree of proliferation, the degree of cytokine production, or thegeneration of cytolytic activity of the T cell population re-exposed inthe antigen. Substantial proliferation of the T cell population inresponse to a particular antigen is considered to be indicative of priorexposure or priming to the antigen.

[0103] Within one aspect of the present invention, a malignancy in whicha HER-2/neu oncogene is associated may be detected. Representativeexamples of such malignancies include breast, ovarian, colon, lung andprostate cancers. An immune response to the HER-2/neu protein, oncegenerated, can be long-lived and can be detected long afterimmunization, regardless of whether the protein is present or absent inthe body at the time of testing. In one embodiment, prior exposure of awarm-blooded animal, such as humans, to the HER-2/neu protein can bedetected by examining for the presence or absence of specific activationof CD4⁺ or CD8⁺ T cells. More specifically, T cells isolated from anindividual by routine techniques (such as by Ficoll/Hypaque densitygradient centrifugation of peripheral blood lymphocytes) are incubatedwith HER-2/neu protein. For example, T cells may be incubated in vitrofor 2-9 days (typically 4 days) at 37° C. with HER-2/neu protein(typically, 5 μg/ml of whole protein or 25 μg/ml of an appropriatepeptide or graded numbers of cells synthesizing HER-2/neu protein). Itmay be desirable to incubate another aliquot of a T cell sample in theabsence of HER-2/neu protein to serve as a control.

[0104] Specific activation of CD4⁺or CD8⁺ T cells may be detected in avariety of ways. Methods for detecting specific T cell activationinclude detecting the proliferation of T cells, the production ofcytokines (e.g., lymphokines), or the generation of cytolytic activity(i.e., generation of cytotoxic T cells specific for HER-2/neu protein).For CD4⁺ T cells, a preferred method for detecting specific T cellactivation is the detection of the proliferation of T cells. For CD8⁺ Tcells, a preferred method for detecting specific T cell activation isthe detection of the generation of cytolytic activity.

[0105] Detection of the proliferation of T cells may be accomplished bya variety of known techniques. For example, T cell proliferation can bedetected by measuring the rate of DNA synthesis. T cells which have beenstimulated to proliferate exhibit an increased rate of DNA synthesis. Atypical way to measure the rate of DNA synthesis is, for example, bypulse-labeling cultures of T cells with tritiated thymidine, anucleoside precursor which is incorporated into newly synthesized DNA.The amount of tritiated thymidine incorporated can be determined using aliquid scintillation spectrophotometer. Other ways to detect T cellproliferation include measuring increases in interleukin-2 (IL-2)production, Ca²⁺ flux, or dye uptake, such as3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Alternatively,synthesis of lymphokines (such as interferon-gamma) can be measured orthe relative number of T cells that can respond to intactp185^(HER-2/neu) protein or peptide may be quantified.

[0106] Intact p185^(HER-2/neu) protein or peptides thereof which arerecognized by cytotoxic T cells may be used within the presentinvention. The peptides may be naturally derived or produced based uponan identified sequence. The peptides for CD8⁺ T cell responses aregenerally 8-10 amino acids in length and the peptides for CD4⁺ T cellresponses are longer, e.g., 15-18 amino acids in length. Peptides forCD8⁺ T cell responses vary according to each individual's class I MHCmolecules. An example of peptides appropriate for CD8⁺ T cell responses(elicited by peptides presented by HLA-A2.1 class I MHC molecules) arepeptides which are 8-10 amino acids in length and contain a leucine atposition 2 and/or a leucine or valine at position 9. Examples ofpeptides (designated by one letter abbreviations for amino acids andfollowed in parentheses by which residues of p185 they correspond)suitable within the present invention for CD8⁺ T cell responses inindividuals that are HLA-A2.1 include peptides consisting essentiallyof: HLYQGCQVV (p48-56) (Seq. ID No. 1); QLFEDNYAL (p106-114) (Seq. IDNo. 26); KIFGSLAFL (p369-377) (Seq. ID No. 27); PLQPEQLQV (p391-399)(Seq. ID No. 2); PLTSIISAV (p650-658) (Seq. ID No. 3); ILLVVVLGV(p661-669) (Seq. ID No. 4); LLVVVLGVV (p662-670) (Seq. ID No. 5);RLLQETELV (p689-697) (Seq. ID No. 6); ILDEAYVMAGV (p767-777) (Seq. IDNo. 28); VMAGVGSPYV (p773-782) (Seq. ID No. 29); CLTSTVQLV (p789-797)(Seq. ID No. 7); DLAARNVLV (p845-853) (Seq. ID No. 8); VLVKSPNHV(p851-859) (Seq. ID No. 9); TLSPGKNGV (p1172-1180) (Seq. ID No. 10);VLGVVFGIL (p666-674) (Seq. ID No. 11); LIKRRQQKI (p674-682) (Seq. ID No.12); KIPVAIKVL (p747-755) (Seq. ID No. 13); ILDEAYVMA (p767-775) (Seq.ID No. 14); QLMPYGCLL (p799-807) (Seq. ID No. 15); QIAKGMSYL (p829-836)(Seq. ID No. 16); LLNWCMQIA (p822-830) (Seq. ID No. 17); RLVHRDLAA(p840-848) (Seq. ID No. 18); DIDETEYHA (p871-879) (Seq. ID No. 19);DLLEKGERL (p933-941) (Seq. ID No. 20); TIDVYMLMV (p948-956) (Seq. ID No.21); MIMVKCWMI (p953-961) (Seq. ID No. 22); DLVDAEEYL (p1016-1024) (Seq.ID No. 23); GLEPSEEEA (p1062-1070) (Seq. ID No. 24); or YLTPQGGAA(p1196-1204) (Seq. ID No. 25).

[0107] Peptides for CD4⁺ T cell responses vary according to eachindividual's class II MHC molecules. Examples of peptides suitablewithin the present invention for CD4⁺ T cell responses include peptidesconsisting essentially of: HLDMLRHLYQGCQVV (p42-56) (Seq. ID No. 30);PLQRLRIVRGTQLFE (p95-109) (Seq. ID No. 31); RLRIVRGTQLFEDNYAL (p98-114)(Seq. ID No. 61); LRSLTEILKGGVLTQ (p142-156) (Seq. ID No. 32);VTYNTDTFESMPNPE (p272-286) (Seq. ID No. 33); NQEVTAEDGTQRCEK (p319-333)(Seq. ID No. 56); TQRCEKCSKPCARVCYGL (p328-345) (Seq. ID No. 60);HLREVRAVTSANIQE (p349-363) (Seq. ID No. 34); VRAVTSANIQEFAGC (p353-367)(Seq. ID No. 35); NQEFAGCKKIFGSL (p360-374) (Seq. ID No. 36);KIFGSLAFLPESFDGD (p369-384) (Seq. ID No. 62); LQVFETLEEITGYLY (p397-411)(Seq. ID No. 58); QVFETLEEITGYLYI (p398-412) (Seq. ID No. 37);QVIRGRILHNGAYSL (p429-443) (Seq. ID No. 57); QECVEECRVLQGLPR (p538-552)(Seq. ID No. 38); ASPLTSIISAVVGIL (p648-662) (Seq. ID No. 59);VVVLGVVFGILIKRR (p664-678) (Seq. ID No. 39); KYTMRRLLQETELVE (p634-698)(Seq. ID No. 40); RRLLQETELVEPLTPS (p688-703) (Seq. ID No. 63);GAMPNQAQMRILKET (p704-718) (Seq. ID No. 41); VKVLGSGAFGTVYKG (p723-737)(Seq. ID No. 42); SPKANKEILDEAYVM (p760-774) (Seq. ID No. 43);GVGSPYVSRLLGICL (p776-790) (Seq. ID No. 44); SRLLGICLTSTVQLV (p783-797)(Seq. ID No. 45); GSQDLLNWCMQIAKG (p818-832) (Seq. ID No. 46);VKITDEGLARLLDID (p859-873) (Seq. ID No. 47); TVWELMTFGAKPYDG (p911-925)(Seq. ID No. 48); PAREIPDLLEKGERL (p927-941) (Seq. ID No. 49);RFRELVSEFSRMARD (p968-982) (Seq. ID No. 50); ELVSEFSRMARDPQ (p971-984)(Seq. ID No. 64); EDDDMGDLVDAEEYL (p10l0-1024) (Seq. ID No. 51);GMGAAKGLQSLPTHD (p1091-1105) (Seq. ID No. 52); TCSPQPEYVNQPDVR(p1132-1146) (Seq. ID No. 53); TLERPKTLSPGKNGV (p1166-1180) (Seq. ID No.54); or GGAVENPEYLTPQGG (p1188-1202) (Seq. ID No. 55).

[0108] It will be evident to those of ordinary skill in the art thatother peptides may be produced for use within the present invention,both for the HLA-A2.1 class I MHC molecule as well as for the otherclass I and class II molecules. A variety of techniques are well knownfor isolating or constructing peptides. Suitable peptides are readilyidentified based upon the disclosure provided herein. Additionalsuitable peptides include those which are longer in length. For example,another peptide has an amino acid sequence corresponding to thatdisclosed in FIG. 1 beginning at about the lysine residue at amino acidposition 676 and extending to about the valine residue at amino acidposition 1255. Such a peptide may be extended (e.g., by the addition ofone or more amino acid residues selected, for example, from position 675to about position 646 of FIG. 1) and/or truncated (e.g., by the deletionof one or more amino acid residues from the carboxyl terminus which isposition 1255 of FIG. 1). Alternatively, suitable peptides may bevariations on other preferred peptides disclosed herein. For example,variations on the peptide designated herein as p650-658 include theextension and/or truncation by the addition or deletion, respectively,of one or more amino acid residues beginning at either position 650 orposition 658 or both positions. As an example, four amino acids areremoved from the amino terminus of p650-658 and four amino acids, suchas the four adjacent to position 658, are added to its carboxylterminus. Although this particular peptide variation results in apeptide with the same number of total amino acids (nine), a peptidevariation on a preferred peptide need not be identical in length.Variations in amino acid sequence that yield peptides havingsubstantially the same desired biological activity are within the scopeof the present invention.

[0109] For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferatein the presence of HER-2/neu protein can be expanded in number either invitro or in vivo. Proliferation of such T cells in vitro may beaccomplished in a variety of ways. For example, the T cells can bere-exposed to HER-2/neu protein. It may be desirable to repeat theexposure of T cells to the HER-2/neu protein to induce proliferation. Itmay be further desirable to include T cell growth factors, such asinterleukin-2, and/or stimulator cells which synthesize HER-2/neuprotein. The addition of stimulator cells is preferred where generatingCD8⁺ T cell responses. HER-2/neu protein-specific T cells can be grownto large numbers in vitro with retention of specificity in response tointermittent restimulation with the immunizing HER-2/neu protein.Briefly, for the primary in vitro stimulation (IVS), large numbers oflymphocytes (e.g., greater than 4×10⁷) are placed in flasks with mediacontaining human serum. HER-2/neu protein (e.g., peptide at 10 μg/ml) isadded directly as well as 5 μg/ml tetanus toxoid. The flasks areincubated at 37° C. for 7 days. For the second IVS, at the end of the 7days, T cells are harvested and placed in new flasks with 2-3×10⁷irradiated peripheral blood mononuclear cells. HER-2/neu protein (e.g.,peptide at 10 μg/ml is added directly). The flasks are incubated at 37°C. for 7 days. On day 2 and day 4 after the second IVS, 2-5 units ofinterleukin-2 (IL-2) is added. For the third IVS, the T cells are placedin wells (e.g., 24 well plates). The T cells are stimulated with theindividual's own EBV transformed B cells coated with the peptide. IL-2is added on days 2 and 4 of each cycle. As soon as the cells are shownto be specific cytotoxic T cells, they are changed to a 10 daystimulation cycle with higher IL-2 (20 units) on days 2, 4 and 6 toexpand them.

[0110] Alternatively, one or more T cells that proliferate in thepresence of HER-2/neu protein can be expanded in number by cloning.Methods for cloning cells are well known in the art. For example, T celllines may be established in vitro and cloned by limiting dilution.Responder T cells are purified from the peripheral blood of sensitizedpatients by density gradient centrifugation and sheep red cell resettingand established in culture by stimulating with the nominal antigen inthe presence of irradiated autologous filler cells. In order to generateCD4⁺ T cell lines, HER-2/neu protein is used as the antigenic stimulusand autologous peripheral blood lymphocytes (PBL) or lymphoblastoid celllines (LCL) immortalized by infection with Epstein Barr virus are usedas antigen presenting cells. In order to generate CD8⁺ T cell lines,autologous antigen-presenting cells transfected with an expressionvector which produces relevant HER-2/neu protein may be used asstimulator cells. Established T cell lines are cloned 2-4 days followingantigen stimulation by plating stimulated T cells at a frequency of 0.5cells per well in 96-well flat-bottom plates with 1×10⁶ irradiated PBLor LCL cells and recombinant interleukin-2 (rIL2) (50 U/ml). Wells withestablished clonal growth are identified at approximately 2-3 weeksafter initial plating and restimulated with appropriate antigen in thepresence of autologous antigen-presenting cells, then subsequentlyexpanded by the addition of low doses of rIL2(10 U/ml) 2-3 daysfollowing antigen stimulation. T cell clones are maintained in 24-wellplates by periodic restimulation with antigen and rIL2 approximatelyevery two weeks.

[0111] Regardless of how an individual's T cells are proliferated invitro, the T cells may be administered to the individual as ananti-cancer composition in an amount effective for therapeutic attackagainst a tumor. Thus, a patient's own T cells (autochthonous T cells)can be used as reagents to mediate specific tumor therapy. Typically,about 1×10⁹ to 1×10¹¹ T cells/M² will be administered intravenously orintracavitary, e.g., in pleural or peritoneal cavities, or in the bed ofa resected tumor. It will be evident to those skilled in the art thatthe number and frequency of administration will be dependent upon theresponse of the patient. Pharmaceutically suitable carriers or diluentsfor T cells include physiological saline or sera. It will be recognizedby one skilled in the art that the composition should be prepared insterile form.

[0112] T cells may also be proliferated in vivo. For example,immunization of an individual with a HER-2/neu peptide (i.e., as avaccine) can induce continued expansion in the number of T cellsnecessary for therapeutic attack against a tumor in which the HER-2/neuoncogene is associated. Typically, about 0.01 μg/kg to about 100 mg/kgbody weight will be administered by the intradermal, subcutaneous orintravenous route. A preferred dosage is about 1 μg/kg to about 1 mg/kg,with about 5 μg/kg to about 200 μg/kg particularly preferred. It will beevident to those skilled in the art that the number and frequency ofadministration will be dependent upon the response of the patient. Itmay be desirable to administer the HER-2/neu peptide repetitively. Itwill be evident to those skilled in this art that more than oneHER-2/neu peptide may be administered, either simultaneously orsequentially. For example, a combination of about 8-15 peptides may beused for immunization. Preferred peptides for immunization are thosethat include all or a portion of the amino acid sequence shown in FIG. 1beginning at about the lysine residue at amino acid position 676 andextending to about the valine residue at amino acid position 1255. Oneor more peptides from other portions of the amino acid sequence shown inFIG. 1 may be added to one or more of the preferred peptides. Neitherintact p185^(HER-2/neu) protein nor a peptide having the amino acidsequence of its entire extracellular domain (i.e., a peptide having anamino acid sequence of the entire amino acid sequence shown in FIG. 1 upto amino acid position 650, plus or minus about one to five positions,and with or without the first 21 amino acid positions) are used alonefor immunization.

[0113] In addition to the HER-2/neu peptide (which functions as anantigen), it may be desirable to include other components in thevaccine, such as a vehicle for antigen delivery and immunostimulatorysubstances designed to enhance the protein's immunogenicity. Examples ofvehicles for antigen delivery include aluminum salts, water-in-oilemulsions, biodegradable oil vehicles, oil-in-water emulsions,biodegradable microcapsules, and liposomes. Examples ofimmunostimulatory substances (adjuvants) includeN-acetylmuramyl-L-alanine-D-isoglutamine (MDP), lipopoly-saccharides(LPS), glucan, IL-12, GM-CSF, gamma interferon and IL-15. It will beevident to those skilled in this art that a HER-2/neu peptide may beprepared synthetically or that a portion of the protein(naturally-derived or synthetic) may be used. When a peptide is usedwithout additional sequences, it may be desirable to couple the peptidehapten to a carrier substance, such as keyhole limpet hemocyanin.

[0114] The present invention also discloses that HER-2/neu protein, inaddition to being immunogenic to T cells, appears to stimulate B-cellsto produce antibodies capable of recognizing HER-2/neu protein.Detection of such antibodies provides another way to diagnose amalignancy in which a HER-2/neu oncogene is associated with themalignancy. Antibodies specific (i.e., which exhibit a binding affinityof about 10⁷ liters/mole or better) for HER-2/neu protein may be foundin a variety of body fluids including sera and ascites. Briefly, a bodyfluid sample is isolated from a warm-blooded animal, such as a human,for whom it is desired to determine whether antibodies specific forHER-2/neu are present. The body fluid is incubated with HER-2/neuprotein under conditions and for a time sufficient to permitimmunocomplexes to form between the protein and antibodies specific forthe protein. For example, a body fluid and HER-2/neu protein may beincubated at 4° C. for 24-48 hours. Following the incubation, thereaction mixture is tested for the presence of immunocomplexes.Detection of one or more immunocomplexes formed between HER-2/neuprotein and antibodies specific for HER-2/neu protein may beaccomplished by a variety of known techniques, such as radioimmunoassays(RIA) and enzyme linked immunosorbent assays (ELISA).

[0115] Suitable immunoassays include the double monoclonal antibodysandwich immunoassay technique of David et al. (U.S. Pat. No.4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al.,in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S.Livingstone, Edinburgh, 1970); the “western blot” method of Gordon etal. (U.S. Pat. No. 4,452,901); immunoprecipitation of labeled ligand(Brown et al., J. Biol. Chem. 255:4980-4983, 1980); enzyme-linkedimmunosorbent assays as described by, for example, Raines and Ross (J.Biol. Chem. 257:5154-5160, 1982); immunocytochemical techniques,including the use of fluorochromes (Brooks et al., Clin. Exp. Immunol.39:477, 1980); and neutralization of activity [Bowen-Pope et al., Proc.Natl. Acad. Sci. USA 81:2396-2400 (1984)], all of which are herebyincorporated by reference. In addition to the immunoassays describedabove, a number of other immunoassays are available, including thosedescribed in U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; 3,935,074;3,984,533; 3,996,345; 4,034,074; and 4,098,876, all of which are hereinincorporated by reference.

[0116] For detection purposes, HER-2/neu protein (“antigen”) may eitherbe labeled or unlabeled. When unlabeled, the antigen find use inagglutination assays. In addition, unlabeled antigen can be used incombination with labeled molecules that are reactive withimmunocomplexes, or in combination with labeled antibodies (secondantibodies) that are reactive with the antibody directed againstHER-2/neu protein, such as antibodies specific for immunoglobulin.Alternatively, the antigen can be directly labeled. Where it is labeled,the reporter group can include radioisotopes, fluorophores, enzymes,luminescers, or dye particles. These and other labels are well known inthe art and are described, for example, in the following U.S. Pat. No.3,766,162; 3,791,932; 3,817,837; 3,996,345; and 4,233,402.

[0117] Typically in an ELISA assay, antigen is adsorbed to the surfaceof a microtiter well. Residual protein-binding sites on the surface arethen blocked with an appropriate agent, such as bovine serum albumin(BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (bufferedsolution of nonfat dry milk which also contains a preservative, salts,and an antifoaming agent). The well is then incubated with a samplesuspected of containing specific antibody. The sample can be appliedneat, or, more often, it can be diluted, usually in a buffered solutionwhich contains a small amount (0.1%-5.0% by weight) of protein, such asBSA, NGS, or BLOTTO. After incubating for a sufficient length of time toallow specific binding to occur, the well is washed to remove unboundprotein and then incubated with an anti-species specific immunoglobulinantibody labeled with a reporter group. The reporter group can be chosenfrom a variety of enzymes, including horseradish peroxidase,beta-galactosidase, alkaline phosphatase, and glucose oxidase.Sufficient time is allowed for specific binding to occur, then the wellis again washed to remove unbound conjugate, and the substrate for theenzyme is added. Color is allowed to develop and the optical density ofthe contents of the well is determined visually or instrumentally.

[0118] In one preferred embodiment of this aspect of the presentinvention, a reporter group is bound to HER-2/neu protein. The step ofdetecting immunocomplexes involves removing substantially any unboundHER-2/neu protein and then detecting the presence or absence of thereporter group.

[0119] In another preferred embodiment, a reporter group is bound to asecond antibody capable of binding to the antibodies specific forHER-2/neu protein. The step of detecting immunocomplexes involves (a)removing substantially any unbound antibody, (b) adding the secondantibody, (c) removing substantially any unbound second antibody andthen (d) detecting the presence or absence of the reporter group. Wherethe antibody specific for HER-2/neu protein is derived from a human, thesecond antibody is an anti-human antibody.

[0120] In a third preferred embodiment for detecting immunocomplexes, areporter group is bound to a molecule capable of binding to theimmunocomplexes. The step of detecting involves (a) adding the molecule,(b) removing substantially any unbound molecule, and then (c) detectingthe presence or absence of the reporter group. An example of a moleculecapable of binding to the immunocomplexes is protein A.

[0121] It will be evident to one skilled in the art that a variety ofmethods for detecting the immunocomplexes may be employed within thepresent invention. Reporter groups suitable for use in any of themethods include radioisotopes, fluorophores, enzymes, luminescers, anddye particles.

[0122] In a related aspect of the present invention, detection ofimmunocomplexes formed between HER-2/neu protein and antibodies in bodyfluid which are specific for HER-2/neu protein may be used to monitorthe effectiveness of cancer therapy for a malignancy in which theHER-2/neu oncogene is associated. Samples of body fluid taken from anindividual prior to and subsequent to initiation of therapy may beanalyzed for the immunocomplexes by the methodologies described above.Briefly, the number of immunocomplexes detected in both samples arecompared. A substantial change in the number of immunocomplexes in thesecond sample (post-therapy initiation)-relative to the first sample(pre-therapy) reflects successful therapy.

[0123] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLES Example 1 CD4+ T Cells Responsive to p185(HER-2/neu) Proteinand Peptides Can Be Detected in Higher Frequency in Patients with BreastCancer than Normal Individuals

[0124] A. p185(HER-2/neu) Protein contains Multiple Segments with AminoAcid Sequences Appropriate for Binding to Class II MHC Molecules

[0125] Soluble proteins are classically processed in the class II MHCpathway. p185^(HER-2/neu) protein is a transmembrane protein present atthe cell surface. When overexpressed, it has been found to be solubleand in the extracellular environment both in vitro and in vivo. In vitrostudies of human breast cancer cell lines found the extracellular domainof p185^(HER-2/neu) in culture media of rapidly growing cells (Alper etal., Cell Growth and Differentiation 1:591-599, 1990; Zabrecky et al.,J. Biol. Chem. 266:1716-1720, 1991). In vivo studies identifiedcirculating portions of the protein in the sera of patients with breastcancer (Leitzel et al., J. Clin. Oncol. 10:1436-1443, 1992; Mori et al.,Jpn. J. Cancer Res. 81:489-494, 1990).

[0126] Peptide segments of the parental HER-2/neu protein with a motifwith theoretical potential to bind to class II MHC molecules wereidentified herein. Locating potential T cell epitopes was aided bycomputer analysis. A protein sequence analysis package, T Sites, thatincorporates several computer algorithms designed to distinguishpotential sites for T cell recognition was used (Feller and de la Cruz,Nature 349:720-721, 1991). Two searching algorithms were used: (1) theAMPHI algorithm described by Margalit (Feller and de la Cruz, Nature349:720-721, 1991; Margalit et al., J. Immunol. 138:2213-2229, 1987)identified epitope motifs according to alpha-helical periodicity andamphipathicity; (2) the Rothbard and Taylor algorithm identified epitopemotifs according to charge and polarity pattern (Rothbard and Taylor,EMBO 7:93-100, 1988). Segments with both motifs are most appropriate forbinding to class II MHC molecules, with the caveat that each particularMHC molecule has a particular binding motif. Using this analysis, morethan 40 potential T cell epitopes in the HER-2/neu protein correspondingto the AMPHI and the Rothbard motifs that would have the potential forbinding to class II MHC molecules were identified (FIG. 1).

[0127] Peptides, each 15 amino acids in length, that encompass both theAMPHI and Rothbard motifs were constructed. The optimal peptide lengthfor class II MHC binding depends upon the particular MHC molecule andmay be shorter than 15 amino acids. However, class II MHC responses toexogenous peptides allow for endocytosis and intracellular processing oflonger peptides. One of the synthetic peptides (p42-56), HLDMLRHLYQGCQVV(Seq. ID No. 30), is located in the extracellular domain and has 33%homology to epidermal growth factor receptor (EGFR) Two other syntheticpeptides, SRLLGICLTSTVQLV (p783-797) (Seq. ID No. 45) andTLERPKTLSPGKNGV (p1166-1180) (Seq. ID No. 54) are both located in theintracellular domain and have 87% and 7% homology to EGFR respectively.The peptides as well as partially purified whole protein(p185^(HER-2/neu)) were used in subsequent defined experiments to detectCD4+T cell proliferation responses (Section C below).

[0128] B. p185(HER-2/neu) Protein can be Obtained and Purified from theHuman Breast Adenocarcinoma Cell Line SKBR3

[0129] Purified p185 for T cell proliferation studies and antibodydetection studies was obtained from the cell line SKBR3. SKBR3 has beenreported on extensively in the literature as a commonly used standardcell line with increased HER-2/neu gene copy number and HER-2/neuprotein overexpression. In one study, SKBR3 cells were found to containa mean HER-2/neu oncogene copy number of 43 copies/cell compared with2.5 copies/cell for MCF-7, a breast cancer cell line considered to be astandard cell line without HER-2/neu gene amplification (Kallionieme etal., Proc. Natl. Acad. Sci. USA 89:5321-5325, 1992). SKBR3 is report-dto be one of the highest known expressors of p185^(HER-2/neu) protein byimmunohistochemistry, 4+ compared to 1+ in MCF-7 (Kerns et al., J.Histochem. & Cytochem. 38:1823-1830, 1990). The same HER-2/neu bands asdescribed in the literature were validated in the present experiments byWestern analysis. Bands detected included p185, p105 (extracellulardomain), and several smaller bands that presumably represent fragmentsof phosphorylated protein (Alper et al., Cell Growth and Differentiation1:591-599, 1990; Zabrecky et al., J. Biol. Chem. 266:1716-1720, 1991;Stern et al., Mol. Cell. Biol. 8:3969-3973, 1988).

[0130] The antibodies used for detecting the HER-2/neu proteinimmunoblotting were commercially prepared by Oncogene Science(Manhasset, N.Y.). The antibody most commonly used in the presentexperiments was c-neu Ab-3; derived by immunization of BALB/c mice witha peptide sequence, TAENPEYLGLDVPV (Seq. ID No. 65), from the carboxyldomain of human c-neu gene product, and fusion of mouse splenocytes withSP2/0 myeloma cells. A second antibody, c-neu AB-1, gave very faintbands when compared with c-neu Ab-3. This antibody was a polyclonalrabbit affinity purified antibody against the peptide sequence,LARLLDIDETEYAD (Seq. ID No. 66), from the kinase domain of the humanc-neu gene product.

[0131] Transmembrane p185^(HER-2/neu) protein was purified from the cellmembrane fraction of SKBR3 by modifications of described methods forother membrane-associated proteins (Dhut et al, Leukemia 4:745-750,1990; Mietzner et al., J. Exp. Med. 165:1041-1057, 1987). Three×10⁶SKBR3 cells were harvested and suspended in phosphate buffered saline(PBS) with the following protease inhibitors; 1 mM PMSF, 1 mMbenzamidine, 5 μg/ml aprotinin. All procedures were done on ice or at 4°C. The cells were then disrupted by sonication at 75 W for a total of 1minute using a high intensity sonifier equipped with a microtip (BransonInstruments, Inc., Stamford, Conn.). The resulting suspension was thencentrifuged for 1 hour at 35,000 rpm to sediment membranes fromcytosolic fraction. The membrane pellet was washed in ice cold PBS withprotease inhibitors and the cycle of sonication/centrifugation wasrepeated twice. All cytosolic (supernatant) and membranous fractionswere tested for the presence of p185^(HER-2/neu) by Western analysis.The protein was noted to be strongly concentrated in the membranefraction.

[0132] Protein concentration of one of these enriched membrane pelletswas determined to be 2625 μg/ml (Protein BioRad assay). p185^(HER-2/neu)is an estimated 8% of membrane protein in SKBR3 (Leitzel et al., J.Clin. Oncol. 10:1436-1443, 1992); therefore, an estimated 210 μg ofp185^(HER-2/neu) were present in the membrane pellet from 3×10⁶ SKBR3cells.

[0133] If desired, the membrane preparation may be further enriched forp185^(HER-2/neu), e.g., by immunoprecipitation. Briefly, 1 μg of c-neu 3antibody and 15 μl protein A agarose were added to the sonicatedmembrane pellet. The mixture was incubated at 4° C. on a rocker for 24hours. The immunoprecipitate was collected by centrifugation in amicro-centrifuge at 2500 rpm for 15 minutes at 4° C., and the resultingpellet was washed several times with PBS, 1% Trition X-100, 0.5% sodiumdeoxycholate, and 0.1% sodium dodecyl sulfate. Silver stain and Westernanalysis showed increased concentration of p185^(HER-2/neu) protein anddecreases in extraneous membrane proteins when compared to membraneenriched pellets alone.

[0134] C. CD4+ T Cells Reactive to p185(HER-2/neu) Protein can beElicited from PBL of Normal Individuals by using an Assay Designed forDetecting Low Frequency Lymphocyte Precursors

[0135] Three assays were used for the detection of CD4⁺ responses: astandard proliferation assay, a screening method for low frequencyevents, and a limiting dilution assay (LDA). Conventional proliferativeassays are capable of readily detecting primed responses. Theproliferative response stimulation index provides a rough correlationwith precursor frequency of antigen-reactive T cells. Any specificproliferative response detected from PBL is considered to be a primedresponse.

[0136] To provide a more quantitative interpretation of CD4⁺ T cellresponses, the assay system developed for detecting low lymphocyteprecursor frequency responses (described below) is used. This assay issimple and cost-effective. In circumstances in which more precision isneeded, the precursor frequency is validated by limiting dilution assays(Bishop and Orosz, Transplantation 47:671-677, 1989).

[0137] Responses greater than detected in normal individuals are definedas a primed response and imply existent immunity. Low responses,detectable only by LDA conditions are considered to be unprimedresponses. An absent response by LDA or a response lower than thatdefined by the normal population analysis is considered to betolerance/anergy.

[0138] In general, primed CD4⁺ T cell responses can be detected inconventional proliferative assays, whereas unprimed responses are notdetectable in the same assays. Detection of small numbers of unprimed Tcells is limited by confounding background thymidine uptake includingthe autologous mixed lymphocyte response (AMLR) to self MHC antigen plusresponses to processed self serum proteins and exogenously added serumproteins.

[0139] To elicit and detect unprimed T cells, an assay system for lowfrequency responses based on Poisson sampling statistics was used (In:Pinnacles, Chiron Corporation, 1:1-2, 1991). This type of analysisapplies specifically to low frequency events in that, if the precursorfrequency is less than the number of cells in one replicate culture,many replicates are required to detect a statistically significantnumber of positives. Theoretically, the analysis will correct forautologous responses by setting up a known positive control (such as PHAor tetanus toxoid) and known negative control (no antigen) andevaluating all data points from lowest to highest irrespective of theexperimental group to which they belong. A cutoff value is calculatedbased on the equation cutoff=M+(F+SD), where M=arithmetic mean, F=3.29,a factor from tables of standardized normal distribution chosen so notmore than 0.1% of the “true negatives” of a normally distributedbackground will be above the cutoff, and SD=standard deviation. In thisscreening assay, wells above the cutoff are considered true positivesthat potentially contain a lymphocyte that is specifically proliferatingto the antigen of interest. Although estimations of lymphocyte precursorfrequency is possible using this method, precise determination requiresformal LDA analysis.

[0140] Analysis of PBL from normal individuals for HER-2/neu peptide andprotein-specific T cells revealed the presence of a low level frequencyof proliferative responses. A representative assay is described in FIG.2. Seven normal subjects were analyzed, 4 males and 3 females. Of theseven individuals evaluated, 57% had a response to whole protein and 29%had a response to at least one individual peptide. The two individualsthat responded to peptide also had responses to parental protein. Threemales and one female had detectable responses to the whole protein. Twomales responded to one of the four peptides. Similar methods can be usedto elicit HER-2/neu reactive T cells from patients with HER-2/neupositive malignancies, but no prior priming in vivo. Alternatively, themethods can be used to assess the efficacy of priming to HER-2/neu invivo and the procurement of immune T cells to be expanded for therapy.

[0141] D. CD4+ T Cells Reactive to p185(HER-2/neu) and Peptide can beDetected in the Peripheral Blood of Patients with HER-2/neu PositiveBreast Cancer in Levels Consistent with a Primed Response

[0142] Four breast cancer patients with known HER-2/neu tumor statushave been evaluated in a standard proliferation assay. Three patientshad tumors which overexpressed the HER-2/neu protein. Proliferation toantigen was consistent with a primed response (FIG. 3) (i.e.,proliferation was detectable in a standard proliferation assay with aStimulation Index (S.I.) greater than 2). One patient was HER-2/neunegative and had a response towards intact HER-2/neu, but no response toHER-2/neu-derived peptides. The patients tested and chosen had differentstages of disease and were in different stages of treatment. Five normalindividuals' responses were analyzed in the same fashion, and none hadan S.I. greater than 2 to any HER-2/neu protein or peptide (Table 1).TABLE 1 (a) Breast Cancer patients HER-2/neu Tetanus p783- p1166-Patient Status Toxoid p185 p42-56 797 1180 A Negative 52 13 2 2 2 BPositive 36 <2 26 19 2 C Positive 7 4 <2 <2 <2 D Positive 10 4 4 5 <2(b) Normal Individuals Tetanus p783- p1166- Normal Toxoid p185 p42-56797 1180 1 6 2 2 <2 <2 2 7 <2 2 <2 <2 3 7 <2 <2 <2 <2 4 10 2 ND ND <2 511 2 ND ND 2

Example 2 CD8⁺ CTL Specific for HER-2/neu Peptides Can Be Generated fromPBL of Normal Individuals by Primary In Vitro Immunization to SyntheticPeptides Derived from the Normal Amino Acid Sequence of p185(HER-2/neu)Protein

[0143] A. p185(HER-2/neu) Protein contains Multiple Segments with anAmino Acid Sequence Motif Appropriate for Binding the Class I MHCMolecule HLA-A2.1

[0144] CD8⁺ T cells recognize peptide bound to class I MHC molecules. Ingeneral, peptide determinants are derived from endogenously synthesizedproteins. The rules which determine the ability of a protein to beprocessed and complexed with class I MHC molecules are not completelyunderstood. Recently, however, it has been determined that peptidesbinding to particular MHC molecules share discernible sequence motifs(Falk et al., Nature 351:290-296, 1991). A peptide motif for binding inthe groove of HLA-A2.1 has been defined by Edman degradation of peptidesstripped from HLA-A2.1 molecules of a cultured cell line (Table 2, fromFalk et al., supra). The method identified the typical or averageHLA-A2.1 binding peptide as being 9 amino acids in length with dominantanchor residues occurring at positions 2 (L) and 9 (V). Commonlyoccurring strong binding residues have been identified at positions 2(M), 4 (E,K), 6 (V), and 8 (K). The identified motif represents theaverage of many binding peptides. TABLE 2 The HLA-A2.1 Restricted MotifPoint Amino Acid Position Assign- 1 2 3 4 5 6 7 8 9 ment DominantBinding L V +3 Anchor Residue Strong Binding M E V K +2 Residue K WeakBinding I A G I I A E L +1 Residue L Y P K L Y S F F D Y T H K P T N M MG Y S V H

[0145] The derived peptide motif as currently defined is notparticularly stringent. Some HLA-A2.1 binding peptides do not containboth dominant anchor residues and the amino acids flanking the dominantanchor residues play major roles in allowing or disallowing binding. Notevery peptide with the current described binding motif will bind, andsome peptides without the motif will bind. However, the current motif isvalid enough to allow identification of some peptides capable ofbinding.

[0146] According to the current motif, the p185^(HER-2/neu) proteincontains a substantial number of peptides with amino acid sequencespossibly appropriate for binding to the class I MHC antigen HLA-A2.1.Evaluation of the 1255 aa structure of p185^(HER-2/neu) revealed atleast 19 peptide segments of 9 aa in length that contained at least oneof the dominant anchor residues. Of note, the current HLA-A2.1 motifplaces 6 amino acids between the dominant anchor amino acids at residues2 and 9. Recent studies show that alterations in secondary structure ofpeptides can sometimes allow for additional intervening residues, andthus longer binding peptides. In the present experiment, 9-mer peptideswere evaluated. The 10 peptides with both dominant residues wereconsidered. The arbitrary scoring system awarded +3 for a dominantanchor residue, +2 for a strong binding residue, and +1 for a weakbinding residue. Emphasis was placed on presence or absence of dominantanchor residues as they appear to be of prime importance for peptidebinding to HLA-A2 (Parker et al., J. Immunol. 148:3580-3587, 1992). Fourpeptides were synthesized (Table 3). One is located in the extracellulardomain of the protein and three are located in the intracellular domain.Homology to EGFR ranges from 11% to 89% (Bargmann et al., Nature319:226-230, 1986). TABLE 3 p185^(HER-2/neu) Peptides Constructed forBinding in HLA-A2.1 Motif p185^(HER-2/neu) Amino Acid Position Peptides1 2 3 4 5 6 7 8 9 Score Location Homology to EGFR p48-56^(HER-2/neu) H LY Q G C Q V V 8* Extracellular 33% (Seq. ID No. 1) p789-797^(HER-2/neu)C L T S T V Q L V 9* Intracellular 89% (Seq. ID No. 7)p851-859^(HER-2/neu) V L V K S P N H V 9* Intracellular 78% (Seq. ID No.9) p1172-1180^(HER-2/neu) T L S P G K N G V 9* Intracellular 11% (Seq.ID No. 10)

[0147] B. Four of Four Peptides with a Motif Theoretically Appropriatefor Binding to HLA-A2.1 can be Shown to Actually Bind to HLA-A2.1 in aClass I MHC Molecule Stabilization Assay

[0148] Having identified and synthesized peptides with a theoreticallikelihood of binding to HLA-A2.1, the constructed peptides wereevaluated as to whether in fact they could bind, the sine quo non ofcytotoxic T lymphocytes (CTL) generation. Of the four peptidesconstructed, all could be shown to bind to HLA-A2 in an assay utilizingthe mutant cell line T2. T2 is a human T-B cell hybrid that has a largehomozygous deletion within the MHC gene region (Riberdy and Cresswell,J. Immunol. 148:2586-2590, 1992; Trousdale et al., Nature 348:741-744,1990; Spies et al., Nature 348:744-747, 1990). The use of T2 todetermine HLA-A2.1 binding peptides has been well defined. T2 does notappropriately process endogenous antigen for presentation with class IMHC molecules. Consequently, cell surface expression of class I MHCmolecules is markedly reduced. However, provision of exogenous peptideswhich bind to and stabilize class I MHC in the presence of B2microglobulin results in increased levels of class I at cell surfacewhich can be easily detected by immunofluorescent staining. T2 withoutexogenous peptide has low expression of HLA-A2 (30%-50%). When incubatedwith peptides able to bind A2, the level of class I MHC stabilizes onthe cell surface and can be measured by immunofluorescent staining.Thus, the T2 line fails to present internal proteins in the class Ipathway, but can bind exogenous peptides, providing that the exogenouspeptides have the appropriate HLA-A2.1 binding motif.

[0149] In this experiment, 1×10⁶ T2 cells were incubated with individualpeptides at a concentration of 25 μg/ml for 18 hours at 37° C. Bindingof peptides to HLA-A2 was determined by immunofluorescent staining witha mouse monoclonal HLA-A2 antibody followed by rabbit antimouse IgG-FITCconjugate. The peptides which bound HLA-A2 increased class I surfaceexpression to 60%-85% (10-15 percentage points over baseline).

[0150] C. CD8⁺ CTL Specific for HER-2/neu p48-56 and p789-797 can beGenerated by Primary In Vitro Immunization

[0151] In general, detection of T cell responses in vitro implies priorpriming has occurred in vivo. It has been difficult and rare to generateCTL in vitro from unprimed populations.

[0152] Conditions for detecting immunity to standard recall antigenswere used and no peptide-specific CTL could be detected. A set ofconditions were derived which have allowed priming to 4 of 4 of thebinding peptides tested to date. The conditions were derived by empiricexperimentation but are consistent with the current paradigm. Conditionsinclude: (1) large numbers of T cells; (2) a concurrent stimulatedprimed CD4⁺ T cell response; (3) IL-2 added late to culture in verysmall amounts; and (4) multiple restimulations.

[0153] Initial experiments examined response to p48-56 which is normallypresent in the extracellular domain and p789-797 which is normallypresent in the intracellular domain, both of which were found to bind toHLA-A2.1. Four of four peptides with a motif theoretically appropriatefor binding to HLA-A2.1 are shown to actually bind to HLA-A2.1 in aclass I MHC molecule stabilization assay (Table 4). T2 cells wereincubated for 18 hours with the depicted synthetic p185^(HER-2/neu)peptides. Cells were then washed and stained with antihuman HLA-A2antibody (3%), a second step FITC-conjugated antibody (3%) was thenadded. The % increase of class I on cell surface as measured byincreased fluorescent intensity of cells incubated with peptide comparedto cells incubated in medium alone is indicated. TABLE 4p185^(HER-2/neu) % Increase of class I Peptides stabilization on T2p48-56 20% p789-797 20% p851-859 12% p1172-1180 10%

[0154] After leukapheresis of a normal homozygous HLA-A2 individual,bulk cultures of lymphocytes (3×10⁷) were incubated with peptide in aconcentration of 10 μg peptide/ml. An individual homozygous for HLA-A2.1was used on the presumption that a double dose of the MHC/peptidecomplex would allow more effective priming. Large numbers of lymphocyteswere used to overcome the presumed low frequency of precursors.Generation of CD8⁺ CTL responses has long been known to requireconcurrent stimulation of CD4⁺ T cell responses to providehelp/amplification. Both peptides used were chosen for class I MHCbinding, and presumably could not stimulate CD4⁺ helper T cells. Toprovide T cell help, low concentrations (5 μg/ml) of tetanus toxoid wereadded to culture along with peptide. So as not to overwhelm or dominatethe culture with the tetanus toxoid response, titrations of tetanustoxoid had previously been assessed in a standard proliferation assaywith the donor's lymphocytes and the concentration of tetanus toxoidthat provided the lowest detectable stimulation index was used.

[0155] Low doses of IL-2 added late to culture were used to maintainlymphocyte proliferation. Within the present disclosure, standardconditions for expanding in vivo primed CTL following secondarysensitization in vitro usually have included IL-2 at 5-10 U/ml on day 2of stimulation. Under primary in vitro immunization conditions, similarconcentrations of IL-2 induced expansion of non-specifically lytic NKand T cells, presumably due to the predominance of NK cells and AMLRresponsive cells relative to peptide-specific CTL. For in vitro priming,the T cell culture received no IL-2 for the first 10 days, with only 1unit/ml administered on day +2 after the second IVS. Thereafter, IL-2 at2 U/ml could be administered on day +2 and day +4 of the 7 daystimulation cycle. T cells were stimulated with peptide on irradiatedPBL as APC every 7 days. Evaluation for specific lytic function wasperformed after the fourth IVS and revealed specific lytic activity butsubstantial non-specific lytic NK and T cell activity. Routine ⁵¹Crrelease assay performed after the tenth IVS (FIG. 4) revealed greaterthan 50% lysis for both bulk T cell lines. Lysis against control targetsof K562 and Daudi was less than 2%.

Example 3 Antibodies Directed Against HER-2/neu Protein Can Be Detectedin the Sera of Patients with Breast Cancer

[0156] A. Antibodies Directed Against p185(HER-2/neu) Protein andp105(HER-2/neu) Extracellular Domain were Detected in the Sera of SomeBreast Cancer Patients

[0157] The sera of 20 patients with breast cancer were analyzed. The 20patients were participants from the Fred Hutchinson Cancer ResearchCenter, Division of Epidemiology WISH study. The patient populationconsisted of women recently diagnosed with breast cancer, generally lessthan 3 months from surgery. Their age was less than 55 and theirHER-2/neu tumor status was unknown. Anti-p185 antibody was found in 55%of the group as evidenced by bands corresponding to the positivecontrol.

[0158] Antibody analysis was based on a modification of standard Westernblotting techniques (Laemmli, Nature 227:680-685, 1970; Burnett, Anal.Biochem. 112:195-203, 1981). A 7.5% SDS polyacrylamide gel was pouredwith a single 12 cm long comb in the stacking gel to create a “trough.”Two immunoprecipitated SKBR3 membrane preparations, described above,were dissolved in loading buffer and layered across the trough. The gelwas then run in standard fashion resulting in a band of equallydistributed proteins across the gel. The protein was transferred tonitrocellulose (Amersham Hybond) for subsequent immunoblotting anddevelopment by chemiluminescence methods (Amersham ECL). Once proteintransfer was complete, the nitrocellulose was cut lengthwise into 25equal strips and placed in a 25 well incubation tray. The nitrocellulosestrips were then blocked with Tris buffered saline and 1% bovine serumalbumin (TBS BSA) for 1 hour. This allows for analysis of 23 patientswith 2 control strips. Patent sera is used as primary antibody, andafter blocking, the strips are incubated for 24 hours at 4° C. with seradiluted 1:200 and 1:400 in TBS BSA. The second antibody is a goatantihuman HRP conjugate which will interact with the chemiluminescentdeveloping reagent (Amersham ECL) resulting in light emission which canbe photographed. A control strip is developed with c-neu Ab3 antibodypreviously described in a similar fashion with this assay both IgA andIgG antibody specific for p185 were detected. Patient sera identifiedthe same p185 band (FIG. 5) as did the known HER-2/neu-specificantibody, providing evidence that some patients have existent antibodyimmunity to HER-2/neu.

[0159] To validate these responses patient sera was tested against amurine cell line (NIH 3T3) that had been transfected with HER-2/neucDNA. As a negative control, untransfected cells were used. Membranepreparations were prepared from the two cell lines and patient sera wasused as primary antibody as previously described. The patient seraidentified the same p185 band as did the known HER-2/neu-specificantibody. That band was present in the cells that contained HER-2/neu,but undetectable in the cells that did not contain HER-2/neu (FIG. 6).

[0160] Recombinant proteins of the extracellular and intracellulardomain portions of HER-2/neu were obtained. The extracellular protein(110 kD) and intracellular protein (75 kD) were resolved on a 7.5%SDS-PAG gel and incubated with patient sera as primary antibody aspreviously described. The sera identified both proteins proving thatsome patients have antibodies directed to both the extracellular andintracellular domain of the HER-2/neu protein (FIG. 7).

[0161] B. Seven Normal Individuals Showed No Evidence of Antibody toHER-2/neu Protein

[0162] In studies to determine the extent to which detection of antibodyto HER-2/neu is specific for malignancy, sera from seven normalindividuals was obtained and analyzed in identical fashion as describedabove. There was no evidence of antibodies directed toward any HER-2/neuprotein.

[0163] C. The Sera of Three Patients with Known HER-2/neu PositiveTumors Contained Antibodies against p185 and p105

[0164] Sera from breast cancer patients whose HER-2/neu tumor status isknown was collected and analyzed to determine the extent to whichantibody to HER-2/neu correlates with the presence of HER-2/neu-positivetumors. Three patients with overexpression of p185^(HER-2/neu) proteinin their primary tumor were analyzed. Antibodies against p185 weredetected in all three. The antibody detected in our studies was IgG.Immunoglobulin class switch from IgM to IgG or IgA require T cell helpoften directed against different epitopes on the same protein molecule.

Example 4 Peptide Based Vaccines Elicit Immunity to HER-2/neu

[0165] A. Materials and Methods

[0166] 1. Animals

[0167] Rats used in this study were Fischer strain 344 (CDF(F-344)/CrlBR) (Charles River Laboratories, Portage Mich.). Animals weremaintained at the University of Washington Animal facilities underspecific pathogen free conditions and routinely used for experimentalstudies between 3 and 4 months of age.

[0168] 2. Antigens

[0169] Nine peptides were constructed, derived from the amino acidsequence of the rat neu protein. The peptides, 15-18 amino acids inlength, were highly homologous to the human HER-2/neu peptide sequence.These peptides were chosen based on an increased probability ofinteraction with human Class II MHC molecules. This theoreticalpotential was evaluated by the use of a protein sequence analysispackage, TSites, that incorporates several computer algorithms designedto distinguish potential sites for T cell recognition (Feller and de laCruz, Nature 349:720-721, 1991). Several peptides identified from therat sequence were predicted to have potential for class II interactionwith both human and murine MHC. Nine peptides were chosen forimmunization of the rats (Table 5). Eight of the nine were in areas of100% homology with human neu. The remaining peptide had greater than 80%homology with human neu (Yamamoto et al., Nature 319:230-234, 1986). Thepeptides were synthesized and purified by H. Zabrowski (University ofWashington, Seattle, Wash.), then dissolved in phosphate-buffered saline(PBS), pH 7.4, to give 2 mg/ml stock solutions. Prior to aliquoting,peptides were sterile filtered, then stored at −70° C. TABLE 5 Peptidesfrom the Rat neu Protein for Immunization Homology Rat Protein to HumanSequence Amino Acids Domain neu p45-59 HLDMLRHLYQGCQVV ECD 100% (Seq. IDNo. 30)  p98-112 PLQRLRIVRGTQLFE ECD 100% (Seq. ID No. 31) p323-337NQEVTAEDGTQRCEK ECD 100% (Seq. ID No. 56) p332-349 TQRCEKCSKPCARVCYGLECD 100% (Seq. ID No. 60) p433-447 RIIRGRILHDGAYSL ECD  80% (Seq. ID No.67) p781-795 GVGSPYVSRLLGICL ICD 100% (Seq. ID No. 44) p788-802SRLLGICLTSTVQLV ICD 100% (Seq. ID No. 45) p932-946 PAREIPDLLEKGERL ICD100% (Seq. ID No. 49) p1171-1185 TLERPKTLSPGKNGV ICD 100% (Seq. ID No.54)

[0170] 3. Immunization

[0171] One group of rats was immunized with a mixture of extracellulardomain (ECD) peptides and one group with a mixture of intracellulardomain (ICD) peptides. The final group received adjuvant alone. Peptideswere administered at a final concentration of 100 μg each in a totalvolume of 200 μl. The animals underwent 3 immunizations each 14-16 daysapart with either CFA or IFA as adjuvant (Sigma ImmunoChemicals, St.Louis, Mo.). 16 days after the third immunization sera was obtained forassessment of immune response.

[0172] 4. Cell Lines

[0173] Two cell lines were used as a source of neu proteins. SKBR3, ahuman breast cancer cell line that is a marked overexpressor ofHER-2/neu (American Type Culture Collection, Rockville, Md.), wasmaintained in culture in 10% fetal bovine serum (FBS) (GeminiBioproducts, Inc., Calabasas, Calif.) and RPMI. DHFR-G8, an NIH/3T3 cellline cotransfected with cneu-p and pSV2-DHFR (American Type CultureCollection, Rockville, Md.), was used as a source of non-transformingrat neu protein (Bernards et al., Proc. Natl. Acad. Sci. USA84:6854-6858, 1987). This cell line was maintained in 10% FBS andDulbecco's modified Eagle's medium with 4.5 g/L glucose. DHFR-G8 cellswere passaged through the same medium supplemented with 0.3 μMmethotrexate at every third passage to maintain the neu transfectant.

[0174] 5. Preparation of Cell Lysates

[0175] Lysates of both SKBR3 and DHFR-G8 were prepared and used as asource of protein for both ELISA and immunoprecipitation studies.Briefly, a lysis buffer consisting of tris base, sodium chloride andTriton-X (1%) pH 7.5 was prepared. Protease inhibitors were added;aprotinin (1μg/ml), benzamidine (1 mM) and PMSF (1 mM). 1 ml of thelysis buffer was used to suspend 10⁷ cells. The cells were vortexed for15 seconds every 10 minutes for an hour until disrupted. All procedureswere performed on ice in a 4° C. cold room. After disruption the cellswere microfuged at 4° C. for 20 minutes. Supernatant was removed fromcell debris and stored in small aliquots at −70° C. until used. Presenceof human and rat neu in the lysates was documented by Western blotanalysis.

[0176] 6. ELISA for rat antibody responses

[0177] 96 well Immulon 4 plates (Baxter SP, Redmond, Wash.: DynatechLaboratories) were incubated overnight at 4° C. with an IgG2a murinemonoclonal antibody directed against rat neu (kindly provided by Dr. M.Green) at a concentration of 10 μg antibody per ml. After incubation,all wells were blocked with PBS and 1% bovine serum albumin (BSA) (SigmaChemical Co., St. Louis, Mo.), 100 μl/well for 4 hours at roomtemperature. The plate was washed with PBS/0.5% Tween and protein wasadded. Rows of wells were coated with alternating PBS/1%BSA and DHFR-G8lysate (10⁸ cells/20 ml PBS), 50 μl/well, overnight at 4° C. Afterwashing, the plate was incubated with rat sera at the followingdilutions: 1:25, 1:50, 1:100, 1:200. The sera was diluted inPBS/1%BSA/1% FBS/25 μg/ml mouse IgG/0.01% NaN3 and then serially intoPBS/1% BSA. 50 μl of diluted sera was added/well and incubated 1 hour atroom temperature. Sheep anti-rat Ig horseradish peroxidase (HRP) wasadded to the wells at a 1:7,500 dilution in PBS/1%BSA and incubated for45 minutes at room temperature (Amersham Co., Arlington Heights, Ill.).Isotype assays were performed similarly with rabbit anti-rat IgG andsheep anti-rat IgM HRP antibodies as the second step antibody at aconcentration of 1:5000 (Serotec Ltd., Oxford, England). Control wellsconsisting of varying dilutions of c-neu-Ab-1, a rabbit polyclonalantibody directed against the kinase portion of human neu which also hasreactivity to rat neu (Oncogene Science, Uniondale, N.Y.), were used asa positive control. These wells received a second step antibody ofgoat-anti rabbit HRP at a 1:5000 dilution (Amersham Co.). Following thefinal wash, TMB (Kirkegaard and Perry Laboratories, Gaithersburg, Md.)developing reagent was added. Color reaction was read at an opticaldensity of 640 nm until the positive control wells reached 0.3 OD. Thereaction was stopped with 1N HCl and the optical density was read at 450nm. The OD of each serum dilution was calculated as the OD of the neucoated wells minus the OD of the PBS/1%BSA coated wells. A pool of 5normal rat sera was run on each plate as a negative control.

[0178] 7. Immunoprecipitation

[0179] Experimental rat sera was used to immunoprecipitate human neufrom the SKBR3 cell line and rat neu from the DHFR-G8 cell line. Acommercially prepared IgGl mouse monoclonal antibody, c-neu-Ab-3, whichcross reacts with both human and rat neu, was used as the positivecontrol antibody in the immunoprecipitation (Oncogene Science). Serafrom 5 pooled normal rats and 2 rats immunized with adjuvant alone andno peptide antigens were used as 2 negative controls. 1 ml of DHFR-G8 orSKBR3 lysate was incubated with 75 μl of rat sera or 10 μl (1 μg) of neuspecific monoclonal antibody and 15 μl of protein A+G (OncogeneScience). The solution was rocked gently overnight at 4° C. After thisincubation, the agarose was pelleted and washed twice in a tris HCl/EDTAbuffer (1M Tris HCl pH 7.5, 0.25 M EDTA, and 5M NaCl), then twice in thesame buffer with NP-40 added to a 0.5% concentration. Theimmunoprecipitates were analyzed by Western blot as described aboveusing c-neu-Ab-1 (Oncogene Science) as the primary antibody. Thisantibody is a neu specific polyclonal rabbit antibody which cross reactswith both human and rat neu.

[0180] 8. ELISA for peptide epitope analysis

[0181] 96 well Immulon 4 plates (Dynatech Laboratories) were incubatedovernight at 4° C. with peptides at a concentration of 10 μg/welldiluted in PBS alternating with rows of PBS/1%BSA. After incubation, allwells were blocked with PBS/1%BSA, 100 μl/well for 4 hours at roomtemperature. The plate was washed with PBS/0.5% Tween. After washing,the plate was incubated with rat sera at the following dilutions: 1:50and 1:100. The sera was diluted in PBS/1%BSA/1% FBS/25 μg/ml mouseIgG/0.01% NaN3 and then serially into PBS/1%BSA. 50 μl of diluted serawas added/well and incubated 1 hour at room temperature. Sheep anti-ratHRP was added to the wells at a 1:7,500 dilution in PBS/1%BSA andincubated for 45 minutes at room temperature. Following the final wash,the TMB developing reagent was added. Color reaction was read at anoptical density of 640 nm until the reading on the most reactive wellreached 0.3 OD. The reaction was stopped with 1N HCl and the opticaldensity was read at 450 nm. The OD of each serum dilution was calculatedas the OD of the peptide coated wells minus the OD of the PBS/1%BSAcoated wells. A pool of 5 normal rat sera was run with each peptide atthe same dilutions as the experimental sera as a negative control.

[0182] 9. Western Blot analysis for rat antibody responses

[0183] Immunoprecipitates of SKBR3 and DHFR-G8 were used as a source ofhuman and rat neu proteins in the Western assays. Recombinant human ECDand ICD (kindly provided by Drs. B. Groner and N. Lydon) were used toevaluate antibody responses to the neu domains. 7.5% polyacrylamide gelswere electrophoresed in the Pharmacia Phast System (Pharmacia LKBBiotechnology AB, Uppsala, Sweden). After transfer to nitrocellulose(Hybond-C, Amersham Co.) the neu proteins were identified by immunoblotin a similar manner. All control blots were developed by using the IgG1mouse monoclonal primary antibody, c-neu-Ab-3 (Oncogene Science). Thisantibody cross reacts with both rat and human neu. The primary antibodywas used in a 1:1000 dilution with tris-buffered saline/1% BSA/0.1%Nonidet P-40. A polyclonal rabbit antimouse HRP-conjugated secondantibody (Amersham Co.) was used in a 1:10,000 dilution. The blot wasthen developed using a chemiluminescent reaction (Amersham ECL).Identically run experimental blots were analyzed with rat sera asprimary antibody. The sera were used in a 1:500 dilution withtris-buffered saline/1% BSA/0.1% Nonidet P-40 in an overnight incubationwith the blot a 4° C. Secondary antibody, goat-anti rat HRP conjugate(Amersham Co.) was used at a 1:5000 dilution. The blots were developedwith ECL detection reagents and exposed to Hyperfilm ECL (Amersham Co.).The film was developed and examined for reaction to human and rat neu aswell as the ICD and ECD domains of the protein. Sera from 5 poolednormal rats and 2 rats immunized with adjuvant alone and no peptideantigens were used as 2 negative controls.

[0184] 10. T Cell Proliferation Assays

[0185] For analysis of neu peptide specific responses: Fresh spleen orlymph node cells were harvested by mechanical disruption and passagethrough wire mesh and washed. 2×10⁵ spleen cells/well and 1×10⁵ lymphnode cells/well were plated into 96-well round bottom microtiter plates(Corning, Corning, NY) with 6 replicates per experimental group. Themedia used consisted of EHAA 120 (Biofluids) with L-glutamine,penicillin/streptomycin, 2-mercaptoethanol, and 5% FBS. Cells wereincubated with 25 μg/ml of the various peptides. The group incubatedwith the peptide mix received 25 μg of each of the peptides. After 4days, wells were pulsed with 1 μCi of [³H]thymidine for 6-8 hours andcounted. Data is expressed as a stimulation index (SI) which is definedas the mean of the experimental wells divided by the mean of the controlwells (no antigen). For analysis of neu protein specific responses:Spleen or lymph node cells were cultured for 3 in vitro stimulations. Atthe time of analysis 1×10⁵ cultured spleen or lymph node T cells wereplated into 96 well microtiter plates as described above. Cells wereincubated with 1μg/ml immunoaffinity column purified rat neu (fromDHFR-G8 cells as the source of rat neu). After 4 days, wells were pulsedwith 1 μCi of [³H]thymidine for 6-8 hours and counted. Data is expressedas a stimulation index which is defined as the mean of the experimentalwells divided by the mean of the control wells (no antigen). 11. Rat TCell Culture

[0186] Spleen and lymph nodes from immunized rats were harvested intosingle cell suspensions. PBMC were isolated by Ficoll/Hypaque densitygradient centrifugation (Histopaque-1083, Sigma Diagnostics, St. Louis,MO). Cells were washed and resuspended in bulk culture of 3×10⁷ cells in6 well plates. The media used consisted of EHAA 120 (Biofluids) withL-glutamine, penicillin/streptomycin, 2-mercaptoethanol, and 10% FBS. Amix of the immunizing peptides were added directly to culture at aconcentration of 10 μg/ml of each peptide. The cultures wererestimulated on the peptide mix every 14 days with syngeneic spleen thathad been preincubated with the peptide mix for 2 hours, irradiated to1000 rads, and then washed. Stimulator to effector ratio was 1:1 in eachculture. After the second week in culture, media was supplemented with50% Con A conditioned media. At the end of 3 in vitro stimulations,cells were >98% CD3+.

[0187] B. Rats Immunized with Peptides Derived from the ICD Portion ofRat neu Protein Develop Antibody Responses to neu Protein

[0188] Rats were immunized with mixtures of either 4 ICD peptides or 5ECD peptides. Following the third immunization, serum and T cells fromimmunized rats were assessed for immunity to neu peptides and protein.Initial experiments assessed rats immunized with ICD peptides forantibody responses to whole neu protein. Serum antibody responses wereanalyzed by ELISA (FIG. 8). The results demonstrate that immunization toICD peptides elicited antibody to whole neu protein. Sera was analyzedat 1:25, 1:50, 1:100, and 1:200 dilution. Results at the 1:25 dilutionare depicted (FIG. 8). neu specific antibody responses titered rapidlyand at a 1:200 dilution the experimental sera demonstrated the samelevel of response as control. Isotype analysis revealed that theantibody responses were predominantly IgG (data not shown).

[0189] C. Rats Immunized with Peptides Derived from the ECD Portion ofRat neu Protein Develop Antibody Responses to neu Protein

[0190] Immunizations with ECD peptides were performed in an identicalfashion as with ICD peptides. ELISA performed on sera from ratsimmunized with ECD peptides revealed the generation of antibodyresponses to whole neu protein (FIG. 9). The responses were equivalentto responses elicited by immunization with ICD peptides. These responseswere predominantly of the IgG subtype (data not shown).

[0191] D. Epitope Analysis of ICD Antibody Responses DemonstratesDominant B cell Epitopes as Well as “Determinant Spreading” betweenDomains

[0192] Mixtures of peptides had been used above for immunization. Todetermine which peptides in the mixture were the predominant B cellepitopes, sera from rats immunized with ICD peptides was analyzed byELISA for responses to individual peptides. Responses to both ICD andECD peptides were evaluated with the presumption that responses to theECD peptides would be non-existent. Results (FIG. 10) revealed differentresponses in each rat. All rats had marked antibody responses to theoverlapping p781 and p788 ICD peptides, although the relative levels ofresponses varied between animals. Responses to p932 and p1171 wereobserved, but were relatively weak. Surprisingly, rats immunized to themixture of ICD peptides displayed significant antibody responses to ECDpeptides. Responses in individual rats varied. Rat 2.2 had substantialresponses to all five ECD peptides evaluated. Rats 2.1 and 2.3 hadweaker responses. Thus, immunization to ICD peptides elicited antibodyresponses to ICD peptides as well as “determinant spreading” with thegeneration of antibody responses to the ECD portion of the molecule.Rats immunized with adjuvant alone did not develop T cell responses toany tested peptide.

[0193] E. Epitope Analysis of ECD Antibody Responses DemonstratesDominant B Cell Epitopes

[0194] Determination of the dominant B cell epitopes in ECD peptideimmunized animals was performed in an identical fashion. Again, therelative responses to individual peptides differed between each animal.Rats immunized with ECD peptides developed substantial responses to p45,p332, and p433 and minimal responses to p98 and p323 (FIG. 11). Thedominant epitope was p45 in rats 1.1 and 1.3, but was p433 in rat 1.2.As with immunization to ECD peptides, determinant spreading wasobserved. All rats developed antibody to p788 in the ICD and rats 1.1and 1.2 responded to p1171. The magnitude and extent of “determinantspreading” appeared to be less in the animals immunized with the ECDpeptides than those immunized with the ICD peptides. However, only alimited number of potential epitopes were examined.

[0195] F. Antibodies Elicited by Immunization to either ICD or ECDPeptides are Specific for and can Immunoprecipitate both Rat neu Proteinand Human HER-2/neu Protein

[0196] The above experiments showed that immunization to neu peptidescould elicit antibody responses to whole rat protein and peptides, asdetermined by ELISA. Verification of the antibody responses to proteinobserved by ELISA was performed by assessing the ability of immune serato immunoprecipitate rat neu protein from lysates of DHFRG-8, an NIH-3T3cell line transfected with non-transforming rat neu. Results showed thatsera from rats immunized with either ECD or ICD peptides couldimmunoprecipitate rat neu (FIG. 12, Panel A).

[0197] The immunizing rat neu peptides were homologous with the humanHER-2/neu protein sequence. Thus, the anti-peptide antibodies elicitedshould be reactive to both rat and human peptides. To determine whetherthe antibodies elicited were also specific for human HER-2/neu protein,experiments evaluated the ability of sera from peptide immunized rats toimmunoprecipitate HER-2/neu from lysates of SKBR3, a human breast cancercell line that overexpresses HER-2/neu. Sera from all rats immunizedwith ICD or ECD peptides could immunoprecipitate HER-2/neu protein whilethe control sera did not (FIG. 12, Panel B).

[0198] G. B Cell Epitopes that are Cross Reactive between Human and Ratneu are Present in both Domains of the Protein

[0199] Antibody elicited by immunization to ICD and ECD peptidesimmunoprecipitated both rat and human neu protein. To further evaluatethe protein domains recognized, sera from rats immunized with ICD andECD peptides was evaluated by Western analysis for reactivity againsthuman recombinant ECD and ICD as well as whole human and rat neuimmunoprecipitated protein. Sera from animals immunized with either ICDor ECD peptides recognized both domains and whole protein from bothspecies (FIG. 13). Control animals had no evidence of antibodiesdirected against either domain. These results verify not only thephenomenon of “determinant spreading” suggested in the peptide epitopeanalysis, but also demonstrate human and rat cross reactive epitopes inboth domains.

[0200] H. Immunization of Rats with ICD Peptides Elicits neuPeptide-Specific T Cell Responses

[0201] The above-detected antibody responses were IgG implying that Tcell help was present and operative in immunoglobulin class switch.Spleen and lymph nodes cells were evaluated for proliferative responsesto the immunizing peptides. Proliferative T cell responses to theimmunizing peptides were observed, but the relative responses betweenindividual rats were varied (FIG. 14). A stimulation index of >2 wasarbitrarily chosen as the cut off of significance. Rat 2.1 did not haveany proliferative response greater than SI of 2 to the mixture ofimmunizing ICD peptides or to individual peptides. Rats 2.2 and 2.3 hadSI>2 to the mixture of ICD peptides with the dominant response to p1171in both rats.

[0202] I. Immunization of Rats with ICD Peptides Elicits neuProtein-Specific T Cell Responses

[0203] Peptide specific T cell lines were derived by repeated in vitrostimulation of spleen cells from peptide immunized mice by a mixture ofthe immunizing peptides. After 40 days the cultured cells were greaterthan 98% CD3+. The cultured T cells from 2 of the 3 immunized ratsdemonstrated substantial responses to protein with SIs of 9 and 16 (FIG.15). The SI from the third rat was >2. No responses to control proteinwere observed.

[0204] J. Immunization of Rats with ECD Peptides Elicits Only WeakPeptide-Specific T Cell Responses

[0205] A similar analysis was performed with T cells derived fromanimals immunized with the ECD peptides. Unlike the responses observedfrom the animals immunized with the mixture of ICD peptides, animalsimmunized with ECD peptides exhibited only weak proliferative responsesto the mixture of ECD peptides as well as to individual peptides (FIG.16). Only one of three rats displayed SI of 2.0 or greater to peptides.

[0206] K. Immunization of Rats with ECD Peptides Elicits Weak, butPositive Responses to neu Protein

[0207] Both splenic and lymph node T cells derived from ECD peptideimmunized rats were analyzed for responses to rat neu protein (FIG. 17).Splenic T cells exhibited low level responses, whereas responses weregreater for lymph node derived T cells. Proliferative responses were notthe same for all animals tested. The maximum SI for spleen derived Tcell lines was 2.1, whereas the maximum SI for lymph node derived Tcells was 3.

[0208] From the foregoing, it will be evident that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A method for the detection of a malignancy in a warm-blooded animal,wherein a HER-2/neu oncogene is associated with the malignancy,comprising the steps of: (a) isolating CD4⁺ T cells from a warm-bloodedanimal; (b) incubating the T cells with HER-2/neu protein; and (c)detecting the presence or absence of specific activation of the T cells,thereby determining the presence or absence of the malignancy.
 2. Themethod of claim 1 wherein a HER-2/neu oncogene is associated with amalignancy selected from the group consisting of breast, ovarian, colon,lung and prostate cancer.
 3. The method of claim 1 wherein the step ofdetecting comprises detecting the presence or absence of proliferationof the T cells.
 4. A method for the detection of a malignancy in awarm-blooded animal, wherein a HER-2/neu oncogene is associated with themalignancy, comprising the steps of: (a) isolating CD8⁺ T cells from awarm-blooded animal; (b) incubating the T cells with HER-2/neu protein;and (c) detecting the presence or absence of specific activation of theT cells, thereby determining the presence or absence of the malignancy.5. The method of claim 4 wherein a HER-2/neu oncogene is associated witha malignancy selected from the group consisting of breast, ovarian,colon, lung and prostate cancer.
 6. The method of claim 4 wherein thestep of detecting comprises detecting the presence or absence ofgeneration of cytolytic activity.
 7. A method for the detection of amalignancy in a warm-blooded animal, wherein a HER-2/neu oncogene isassociated with the malignancy, comprising the steps of: (a) contactinga body fluid, suspected of containing antibodies specific for HER-2/neuprotein, with HER-2/neu protein; (b) incubating the body fluid underconditions and for a time sufficient to allow immunocomplexes to form;and (c) detecting the presence or absence of immunocomplexes formedbetween the HER-2/neu protein and antibodies in the body fluid specificfor the HER-2/neu protein, thereby determining the presence or absenceof the malignancy.
 8. The method of claim 7 wherein a HER-2/neu oncogeneis associated with a malignancy selected from the group consisting ofbreast, ovarian, colon, lung and prostate cancer.
 9. The method of claim7 wherein a reporter group is bound to a second antibody capable ofbinding to the antibodies, and wherein the step of detecting comprises(a) removing substantially any unbound antibody, (b) adding the secondantibody, (c) removing substantially any unbound second antibody, and(d) detecting the presence or absence of the reporter group.
 10. Themethod of claim 9 wherein the second antibody is an anti-human antibody.11. The method of claim 9 wherein the reporter group is selected fromthe group consisting of radioisotopes, fluorophores, enzymes,luminescers, and dye particles.
 12. The method of claim 7 wherein areporter group is bound to a molecule capable of binding to theimmunocomplexes, and wherein the step of detecting comprises (a) addingthe molecule, (b) removing substantially any unbound molecule, and (c)detecting the presence or absence of the reporter group.
 13. The methodof claim 12 wherein the molecule capable of binding to theimmunocomplexes is protein A.
 14. The method of claim 12 wherein thereporter group is selected from the group consisting of radioisotopes,fluorophores, enzymes, luminescers, and dye particles.
 15. The method ofclaim 7 wherein a reporter group is bound to the HER-2/neu protein, andwherein the step of detecting comprises removing substantially anyunbound HER-2/neu protein and thereafter detecting the presence orabsence of the reporter group.
 16. The method of claim 15 wherein thereporter group is selected from the group consisting of radioisotopes,fluorophores, enzymes, luminescers, and dye particles.
 17. A method formonitoring the effectiveness of cancer therapy-sin a warm-blooded animalwith a malignancy, wherein a HER-2/neu oncogene is associated with themalignancy, comprising the steps of: (a) contacting a first body fluidsample, taken from the warm-blooded animal prior to initiation oftherapy, with HER-2/neu protein; (b) incubating the body fluid underconditions and for a time sufficient to allow immunocomplexes to form;(c) detecting immunocomplexes formed between the HER-2/neu protein andantibodies in the body fluid specific for the HER-2/neu protein; (d)repeating steps (a), (b), and (c) on a second body fluid sample takenfrom the animal subsequent to the initiation of therapy; and (e)comparing the number of immunocomplexes detected in the first and secondbody fluid samples, thereby monitoring the effectiveness of the therapyin the animal.
 18. The method of claim 17 wherein a HER-2/neu oncogeneis associated with a malignancy selected from the group consisting ofbreast, ovarian, colon, lung and prostate cancer.
 19. The method ofclaim 17 wherein a reporter group is bound to a second antibody capableof binding to the antibodies, and wherein the step of detectingcomprises (a) removing substantially any unbound antibody, (b) addingthe second antibody, (c) removing substantially any unbound secondantibody, and (d) detecting the presence or absence of the reportergroup.
 20. The method of claim 19 wherein the second antibody is ananti-human antibody.
 21. The method of claim 19 wherein the reportergroup is selected from the group consisting of radioisotopes,fluorophores, enzymes, luminescers, and dye particles.
 22. The method ofclaim 17 wherein a reporter group is bound to a molecule capable ofbinding to the immunocomplexes, and wherein the step of detectingcomprises (a) adding the molecule, (b) removing substantially anyunbound molecule, and (c) detecting the presence or absence of thereporter group.
 23. The method of claim 22 wherein the molecule capableof binding to the immunocomplexes is protein A.
 24. The method of claim22 wherein the reporter group is selected from the group consisting ofradioisotopes, fluorophores, enzymes, luminescers, and dye particles.25. The method of claim 17 wherein a reporter group is bound to theHER-2/neu protein, and wherein the step of detecting comprises removingsubstantially any unbound HER-2/neu protein and thereafter detecting thepresence or absence of the reporter group.
 26. The method of claim 25wherein the reporter group is selected from the group consisting ofradioisotopes, fluorophores, enzymes, luminescers, and dye particles.27. A method for treating a malignancy in a warm-blooded animal, whereina HER-2/neu oncogene is associated with the malignancy, comprising thesteps of: (a) isolating CD4⁺ T cells from a warm-blooded animal; (b)incubating the T cells in the presence of HER-2/neu protein, such thatthe T cells proliferate; and (c) administering to the warm-bloodedanimal an effective amount of the proliferated T cells.
 28. The methodof claim 27 wherein a HER-2/neu oncogene is associated with a malignancyselected from the group consisting of breast, ovarian, colon, lung andprostate cancer.
 29. The method of claim 27 wherein the step ofincubating the T cells is repeated one or more times.
 30. A method fortreating a malignancy in a warm-blooded animal, wherein a HER-2/neuoncogene is associated with the malignancy, comprising the steps of: (a)isolating CD4⁺ T cells from a warm-blooded animal; (b) incubating the Tcells in the presence of HER-2/neu protein, such that the T cellsproliferate; (c) cloning one or more cells that proliferated in thepresence of HER-2/neu protein; and (d) administering to the warm-bloodedanimal an effective amount of the cloned T cells.
 31. The method ofclaim 30 wherein a HER-2/neu oncogene is associated with a malignancyselected from the group consisting of breast, ovarian, colon, lung andprostate cancer.
 32. A method for treating a malignancy in awarm-blooded animal, wherein a HER-2/neu oncogene is associated with themalignancy, comprising the steps of: (a) isolating CD8⁺ T cells from awarm-blooded animal; (b) incubating the T cells in the presence ofHER-2/neu protein, such that the T cells proliferate; and (c)administering to the warm-blooded animal an effective amount of theproliferated T cells.
 33. The method of claim 32 wherein a HER-2/neuoncogene is associated with a malignancy selected from the groupconsisting of breast, ovarian, colon, lung and prostate cancer.
 34. Themethod of claim 32 wherein the step of incubating the T cells isrepeated one or more times.
 35. A method for treating a malignancy in awarm-blooded animal, wherein a HER-2/neu oncogene is associated with themalignancy, comprising the steps of: (a) isolating CD8⁺ T cells from awarm-blooded animal; (b) incubating the T cells in the presence ofHER-2/neu protein, such that the T cells proliferate; (c) cloning one ormore cells that proliferated in the presence of HER-2/neu protein; and(d) administering to the warm-blooded animal an effective amount of thecloned T cells.
 36. The method of claim 35 wherein a HER-2/neu oncogeneis associated with a malignancy selected from the group consisting ofbreast, ovarian, colon, lung and prostate cancer.
 37. A method fortreating a malignancy in a warm-blooded animal, wherein a HER-2/neuoncogene is associated with the malignancy, comprising immunizing theanimal with a HER-2/neu peptide recognized by T cells, said peptide notbeing the extracellular domain of the protein expression product of aHER-2/neu oncogene.
 38. The method of claim 37 wherein a HER-2/neuoncogene is associated with a malignancy selected from the groupconsisting of breast, ovarian, colon, lung and prostate cancer.
 39. Themethod of claim 37 wherein the step of immunizing comprisesadministering the HER-2/neu peptide repetitively to the animal.
 40. Ananti-cancer therapeutic composition, comprising T cells proliferated inthe presence of HER-2/neu protein, in combination with apharmaceutically acceptable carrier or diluent.
 41. A peptide consistingessentially of: His-Leu-Tyr-Gln-Gly-Cys-Gln-Val-Val (Seq. ID No. 1);Pro-Leu-Gln-Pro-Glu-Gln-Leu-Gln-Val (Seq. ID No. 2);Pro-Leu-Thr-Ser-Ile-Ile-Ser-Ala-Val (Seq. ID No. 3);Ile-Leu-Leu-Val-Val-Val-Leu-Gly-Val (Seq. ID No. 4);Leu-Leu-Val-Val-Val-Leu-Gly-Val-Val (Seq. ID No. 5);Arg-Leu-Leu-Gln-Glu-Thr-Glu-Leu-Val (Seq. ID No. 6);Cys-Leu-Thr-Ser-Thr-Val-Gln-Leu-Val (Seq. ID No. 7);Asp-Leu-Ala-Ala-Arg-Asn-Val-Leu-Val (Seq. ID No. 8);Val-Leu-Val-Lys-Ser-Pro-Asn-His-Val (Seq. ID No. 9);Thr-Leu-Ser-Pro-Gly-Lys-Asn-Gly-Val (Seq. ID No. 10);Val-Leu-Gly-Val-Val-Phe-Gly-Ile-Leu (Seq. ID No. 11);Leu-Ile-Lys-Arg-Arg-Gln-Gln-Lys-Ile (Seq. ID No. 12);Lys-Ile-Pro-Val-Ala-Ile-Lys-Val-Leu (Seq. ID No. 13);Ile-Leu-Asp-Glu-Ala-Tyr-Val-Met-Ala (Seq. ID No. 14);Gln-Leu-Met-Pro-Tyr-Gly-Cys-Leu-Leu (Seq. ID No. 15);Gln-Ile-Ala-Lys-Gly-Met-Ser-Tyr-Leu (Seq. ID No. 16);Leu-Leu-Asn-Trp-Cys-Met-Gln-Ile-Ala (Seq. ID No. 17);Arg-Leu-Val-His-Arg-Asp-Leu-Ala-Ala (Seq. ID No. 18);Asp-Ile-Asp-Slu-Thr-Glu-Tyr-His-Ala (Seq. ID No. 19);Asp-Leu-Leu-Glu-Lys-Gly-Glu-Arg-Leu (Seq. ID No. 20);Thr-Ile-Asp-Val-Tyr-Met-Leu-Met-Val (Seq. ID No. 21);Met-Ile-Met-Val-Lys-Cys-Trp-Met-Ile (Seq. ID No. 22);Asp-Leu-Val-Asp-Ala-Glu-Glu-Tyr-Leu (Seq. ID No. 23);Gly-Leu-Glu-Pro-Ser-Glu-Glu-Glu-Ala (Seq. ID No. 24); orTyr-Leu-Thr-Pro-Gln-Gly-Gly-Ala-Ala (Seq. ID No. 25).
 42. A peptideconsisting essentially of:His-Leu-Asp-Met-Leu-Arg-His-Leu-Tyr-Gln-Gly-Cys-Gln-Val-Val (Seq. ID No.30); Pro-Leu-Gln-Arg-Leu-Arg-Ile-Val-Arg-Gly-Thr-Gln-Leu-Phe-Glu (Seq.ID No. 31); Leu-Arg-Ser-Leu-Thr-Glu-Ile-Leu-Lys-Gly-Gly-Val-Leu-Ile-Gln(Seq. ID No. 32);Val-Thr-Tyr-Asn-Thr-Asp-Thr-Phe-Glu-Ser-Met-Pro-Asn-Pro-Glu (Seq. ID No.33); His-Leu-Arg-Glu-Val-Arg-Ala-Val-Thr-Ser-Ala-Asn-Ile-Gln-Glu (Seq.ID No. 34); Val-Arg-Ala-Val-Thr-Ser-Ala-Asn-Ile-Gln-Glu-Phe-Ala-Gly-Cys(Seq. ID No. 35);Asn-Ile-Gln-Glu-Phe-Ala-Gly-Cys-Lys-Lys-Ile-Phe-Gly-Ser-Leu (Seq. ID No.36); Gln-Val-Phe-Glu-Thr-Leu-Glu-Glu-Ile-Thr-Gly-Tyr-Leu-Tyr-Ile (Seq.ID No. 37); Gln-Glu-Cys-Val-Glu-Glu-Cys-Arg-Val-Leu-Gln-Gly-Leu-Pro-Arg(Seq. ID No. 38);Val-Val-Val-Leu-Gly-Val-Val-Phe-Gly-Ile-Leu-Ile-Lys-Arg-Arg (Seq. ID No.39); Lys-Tyr-Thr-Met-Arg-Arg-Leu-Leu-Gln-Glu-Thr-Glu-Leu-Val-Glu (Seq.ID No. 40); Gly-Ala-Met-Pro-Asn-Gln-Ala-Gln-Met-Arg-Ile-Leu-Lys-Glu-Thr(Seq. ID No. 41);Val-Lys-Val-Leu-Gly-Ser-Gly-Ala-Phe-Gly-Thr-Val-Tyr-Lys-Gly (Seq. ID No.42); Ser-Pro-Lys-Ala-Asn-Lys-Glu-Ile-Leu-Asp-Glu-Ala-Tyr-Val-Met (Seq.ID No. 43); Gly-Val-Gly-Ser-Pro-Tyr-Val-Ser-Arg-Leu-Leu-Gly-Ile-Cys-Leu(Seq. ID No. 44);Ser-Arg-Leu-Leu-Gly-Ile-Cys-Leu-Thr-Ser-Thr-Val-Gln-Leu-Val (Seq. ID No.45); Gly-Ser-Gln-Asp-Leu-Leu-Asn-Trp-Cys-Met-Gln-Ile-Ala-Lys-Gly (Seq.ID No. 46); Val-Lys-Ile-Thr-Asp-Phe-Gly-Leu-Ala-Arg-Leu-Leu-Asp-Ile-Asp(Seq. ID No. 47);Thr-Val-Trp-Glu-Leu-Met-Thr-Phe-Gly-Ala-Lys-Pro-Tyr-Asp-Gly (Seq. IDNo.48); Pro-Ala-Arg-Glu-Ile-Pro-Asp-Leu-Leu-Glu-Lys-Gly-Glu-Arg-Leu(Seq. ID No. 49);Arg-Phe-Arg-Glu-Leu-Val-Ser-Glu-Phe-Ser-Arg-Met-Ala-Arg-Asp (Seq. ID No.50); Glu-Asp-Asp-Asp-Met-Gly-Asp-Leu-Val-Asp-Ala-Glu-Glu-Tyr-Leu (Seq.ID No. 51); Gly-Met-Gly-Ala-Ala-Lys-Gly-Leu-Gln-Ser-Leu-Pro-Thr-His-Asp(Seq. ID No. 52);Thr-Cys-Ser-Pro-Gln-Pro-Glu-Tyr-Val-Asn-Gln-Pro-Asp-Val-Arg (Seq. ID No.53); Thr-Leu-Glu-Arg-Pro-Lys-Thr-Leu-Ser-Pro-Gly-Lys-Asn-Gly-Val (Seq.ID No. 54); Gly-Gly-Ala-Val-Glu-Asn-Pro-Glu-Tyr-Leu-Thr-Pro-Gln-Gly-Gly(Seq. ID No. 55);Asn-Gln-Glu-Val-Thr-Ala-Glu-Asp-Gly-Thr-Gln-Arg-Cys-Glu-Lys (Seq. ID No.56); Gln-Val-Ile-Arg-Gly-Arg-Ile-Leu-His-Asn-Gly-Ala-Tyr-Ser-Leu (Seq.ID No. 57); Leu-Gln-Val-Phe-Glu-Thr-Leu-Glu-Glu-Ile-Thr-Gly-Tyr-Leu-Tyr(Seq. ID No. 58);Ala-Ser-Pro-Leu-Thr-Ser-Ile-Ile-Ser-Ala-Val-Val-Gly-Ile-Leu (Seq. ID No.59);Thr-Gln-Arg-Cys-Glu-Lys-Cys-Ser-Lys-Pro-Cys-Ala-Arg-Val-Cys-Tyr-Gly-Leu(Seq. ID No. 60);Arg-Leu-Arg-Ile-Val-Arg-Gly-Thr-Gln-Leu-Phe-Glu-Asp-Asn-Tyr-Ala-Leu(Seq. ID No. 61);Lys-Ile-Phe-Gly-Ser-Leu-Ala-Phe-Leu-Pro-Glu-Ser-Phe-Asp-Gly-Asp (Seq. IDNo. 62); Arg-Arg-Leu-Leu-Gln-Glu-Thr-Glu-Leu-Val-Glu-Pro-Leu-Thr-Pro-Ser(Seq. ID No. 63); orGlu-Leu-Val-Ser-Glu-Phe-Ser-Arg-Met-Ala-Arg-Asp-Pro-Gln (Seq. ID No.64).
 43. A peptide consisting essentially of the amino acid sequence ofFIG. 1 from lysine, amino acid 676, to valine, amino acid 1255.